0656 v3.0 Duolink In Situ Brightfield User Manual
Contents
1. gt gt Proceed to the Detection protocol in section 73 on page 18 Duolink In Sltu Brightfield 7 3 DETECTION PROTOCOL Proceed from step 4e of the PLA probe protocols Use open droplet reactions without a cover slip and perform all incubations in a humidity chamber The volume examples are based on 40 yl reaction volume suitable for 1 cm reaction area Adjust the volumes corresponding to your specific delimited reaction area see the Reaction Volume Guide Art no 80520 or section 6 4 5 LIGATION a Dilute the Ligation stock 1 5 in high purity water and mix Wait to add the Ligase until immediately before addition to the samples Take the addition of Ligase into account when calculating the amount of water added E g for a 40 ul reaction take 8 ul of the 5x Ligation stock and 31 ul of high purity water b Tap off the PLA probe solution from the slides c Wash the slides in 1x Wash Buffer A for 2 x 5 min under gentle agitation d Add Ligase to the Ligation solution from step a at a 1 40 dilution and vortex E g for a 40 ul reaction add 1ul of Ligase to 39 ul of Ligation solution e Add the Ligation Ligase solution to each sample f Incubate the slides in a pre heated humidity chamber for 30 min at 37 C 6 AMPLIFICATION a Dilute the Duolink In Situ Amplification stock 1 5 in high purity water and mix Wait to add the Polymerase until immediately before addition to the sample Take the addition2. 6 4 REACTION VOLUME gt Use open droplet reactions b Perform the incubations without a cover slip b Perform all incubations in a pre heated humidity chamber Use volumes corresponding to your delimited reaction area Never use less than 15 ul of total reaction volume see Table 1 or Reaction Volume Guide Art no 80520 Note t is important that all incubations are performed in a humid environment to prevent excessive evaporation If the sample goes dry this will give rise to severe artifacts Table 1 Suitable reaction volume for different reaction areas AREA TOTAL REACTION VOLUME 0 2 cm 1 cm 2 cm S ism 4cm 6cm 8cm 10 cm 15 ul 40 ul 80 ul 120 ul 160 ul 240 ul 320 ul 400 ul Duolink In Situ Brightfield 15 7 Assay protocol 7 1 PLA PROBE PROTOCOL CUSTOM SOLUTIONS Follow this protocol if you use your own blocking solution and antibody diluent that you know work with your primary antibodies Before you start your samples should be deposited on glass slides and pre treated with respect to fixation retrieval and or permeabilization Use open droplet reactions without a cover slip and perform all incubations in a humidity chamber Use volumes corresponding to your delimited reaction area see the Reaction Volume Guide Art no 80520 or section 6 4 To avoid dilution of the reagents tap off as much wash buffer as possible without letting the sample dry 1 PEROXIDASE QUENCHING
3. Some general guidelines are given below PROBLEM PROBABLE CAUSE SUGGESTED SOLUTION No or few No or insufficient Check primary antibodies in an ordinary IHC or IF against a positive signals in binding of control in relevant samples with similar fixation retrieval protocol positive primary blocking and buffer conditions as used for PLA Evaluate and optimize samples antibodies fixative retrieval incubation temperature time concentration and buffer composition Insufficient Ensure that your reaction area corresponds to the reaction volume reaction volume The droplet must cover the reaction area Encircle your reaction area using a hydrophobic barrier and incubate the slides in a humidity chamber to prevent evaporation Do NOT use a cover slip to disperse the droplet No amplification Ensure that the Polymerase has been stored at 20 C Check that the incubator is not warmer than 37 C Inefficient Keep ligation incubation time and temperature Ensure that no ligation excessive amount of wash solution remain on slide before addition of ligation reagents Ensure that the Ligase is active i e has been kept at 20 C and that correct dilution of the reagents have been used Prepare fresh dilutions just before use do not allow mix with enzyme to stand for more than 5 minutes before use Inefficient If the signals are very weak they can appear to be few as only a amplification fraction reaches above detection thresh
4. a Add the Hydrogen Peroxide solution to each sample Note Optimization of incubation time may be needed b Incubate at room temperature for 5 min c Wash the slides with 1x Wash Buffer A for 2 x 5 min under gentle agitation 2 BLOCKING Use your previously tested blocking solution a Add blocking solution to each sample b Incubate the slides 2 PRIMARY ANTIBODIES Use your previously tested buffer to dilute your primary antibodies a Dilute your primary antibody to a suitable concentration in your custom antibody diluent If using two primary antibodies dilute the two antibodies in the same diluent b Tap off the blocking solution from the slides Try to obtain an equal residual volume on each slide as this will affect reproducibility Do not allow the samples to dry before adding the primary antibodies as this will cause background c Add the primary antibody solution to each sample d Incubate in a humidity chamber Use temperature and time optimal for your primary antibodies 4 PLA PROBES Use the same buffer as for your primary antibodies or use Duolink In Situ Antibody Diluent Note The buffer should contain the blocking agent that was used for blocking the samples a Mix and dilute the two PLA probes 1 5 in your chosen buffer Allow the mixture to sit for 20 min at room temperature E g for a 40 ul reaction take 8 ul of PLA probe MINUS stock 8 ul of PLA probe PLUS stock and 24 ul of the antibody buff
5. Brightfield 3 4 1 Introduction Duolink In Situ reagents from Olink Bioscience enable detection visualization and quantification of individual proteins protein modifications and protein interactions in tissue and cell samples prepared for microscopy The target is detected using one or two primary antibodies depending on the application In the case that two primary antibodies are used they must have been raised in different species The Duolink In Situ reagents are based on in situ PLA which is a proximity ligation assay technology A pair of oligonucleotide labeled secondary antibodies PLA probes generates a signal only when the two PLA probes have bound in close proximity either to the same primary antibody or two primary antibodies that have bound to the sample in close proximity The signal from each detected pair of PLA probes is visualized as an individual spot These PLA signals can be quantified counted and assigned to a specific subcellular location based on microscopy images Duolink In Situ Brightfield 2 Principle of the assay Typical starting materials are adherent cells cytospin preparations or tissue sections on a glass slide fixed pre treated and blocked with a blocking reagent according to the reguirements of the primary antibodies used A The samples are incubated with primary antibodies that bind to the protein s to be detected B Secondary antibodies conjugated with oligonucleotides
6. PLA probe MINUS and PLA probe PLUS are added to the reaction and incubated C The Ligation solution consisting of two oligonucleotides illustrated as red bands and Ligase is added and the oligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity D The Amplification solution consisting of nucleotides not shown is added together with Polymerase yellow The oligonucleotide arm of one of the PLA probes acts as a primer for a rolling circle amplification RCA reaction using the ligated circle as a template generating a concatemeric repeated sequence product extending from the oligonucleotide arm of the PLA probe E The Detection solution consisting of horseradish peroxidase HRP labeled oligonucleotides is added and the labeled oligonucleotides will hybridize to the RCA product The signal is easily made visible as a distinct brown dot after enzymatic conversion of NovaRED substrate and analyzed by brightfield microscopy Fig 1 Assay principle Duolink In Sltu Brightfield 5 3 Applications 3 1 DETECT AND OUANTIFY PROTEIN INTERACTIONS Detection and guantification of interacting proteins can be done using two different primary antibodies against each of the two proteins of interest 3 2 DETECT AND OUANTIFY PROTEIN MODIFICATION Detection and guantification of a protein and its specific post translational modification can be done using two differen
7. dry well before analyzing in a brightfield microscope using at least a 20x objective Duolink In Sltu Brightfield 8 Results 8 1 TYPICAL RESULTS The result from using Duolink In Situ Detection Reagents Brightfield is typically a number of distinct red brown spots PLA signals in the tissue see Figure 10 In some cases when studying highly expressed proteins the density of signals may be so high that the signals coalesce see Figure 11 aur 4 2 4 07 ANY 7 are s E e sa A P N e Z 2955 e dee e SN Ps T Tr S E Gy se ae p ee 9 0 S t i 4 e gee meta Ca Fig 10 Detection of HER2 HER3 heterodimers in FFPE breast cancer tissue using Duolink In Situ with two primary antibodies where the primary antibodies have been titrated to the optimal concentration to give individual signals PLA signals are shown in red brown and the nuclei in blue A Positive sample B Negative control no primary antibodies Fig 11 Detection of the HER2 receptor in FFPE breast cancer tissue scored as 24 with regard to HER2 with IHC by Pantomics Inc using Duolink In Situ with one primary antibody where the primary antibody needs to be titrated to give individual signals PLA signals are shown in red brown and the nuclei in blue A Positive sample B Negative control no primary antibody 20 Duolink In Situ Brightfield 8 2 IMAGE ACOUISITION The PLA signal detected with Duolink In Situ Detection
8. of the sample if you have not already optimized your primary antibody with another blocking solution Supplied in ready to use concentration b Antibody Diluent For dilution of PLA probes and in some cases the primary antibodies section 5 4 3 Supplied in ready to use concentration b PLA probe 5x stock Secondary antibody conjugated with a PLA oligonucleotide 4 1 2 DUOLINK IN SITU DETECTION REAGENTS BRIGHTFIELD Each Duolink In Situ Detection Reagents Brightfield contains two boxes A and B together they contain the following b Ligation 5x Contains oligonucleotides that hybridize to the PLA probes and all components needed for ligation except the Ligase Ligase 1 U ul gt Amplification bx All components needed for Rolling Circle Amplification RCA except the Polymerase Polymerase 10 U ul Detection Brightfield bx HRP labeled oligonucleotides Hydrogen peroxide NovaRED substrate Nuclear stain Mayers hematoxylin v v v v v Duolink In Sltu Brightfield 7 4 1 3 DUOLINK IN SITU BRIGHTFIELD MOUNTING MEDIUM A non agueous xylene based and permanent hard set mounting medium 4 1 4 DUOLINK IN SITU WASH BUFFER A Duolink In Situ Wash Buffer A contains pouches to be dissolved in high purity water 4 2 REAGENTS TO BE SUPPLIED BY THE USER b Reagents required for fixation and antigen retrieval of the sample according to your own protocol specific for each antigen and antibody used Primary
9. than the heat induced epitope retrieval does Note Slides developed with NovaRED should be dehydrated and mounted with a non aqueous mounting media e g Duolink In Situ Brightfield Mounting Media Xylene based 5 5 2 NUCLEAR STAINING Depending on how strong your HRP NovaRED staining is you can choose how strong the nuclear staining should be However if you intend to use image analysis we can recommend the nuclei to be well stained After staining for 2 min in Duolink In Situ Nuclear stain the specimen needs to be left in running tap water for about 10 min This step is necessary to give the nuclear stain a chance to mature and to bring forth its specific blue color Please note that you will achieve the best result with running tap water not only to leave the slides in standing tap water Duolink In Situ Brightfield 5 5 3 DEHYDRATION AND CLEARING Dehydration is performed in steps of incubations in baths of increasing alcohol percentage or possibly other dehydration liquids Prior to the first bath the slides are left in distilled deionized or high purity water Pass the slides through the alcohol gradient for three minutes in each bath according to the User Manual using two baths for each alcohol step When transferred to your first clearing bath let stand for 10 minutes before changing to the mounting bath Duolink In Situ Detection Reagents Brightfield has been evaluated with alcohol as dehydration steps and xylene as clearing
10. the sample Ligation 5x store at 20 C gt Thaw at room temperature and vortex before use b Dilute 1 5 in high purity water immediately before use Note The buffer contains DTT that may precipitate during freezing Vortex to dissolve homogenize Ligase 1 U ul store at 20 C The Ligase should be kept at 20 C at all times Use a freezing block 20 C when removing the enzyme from the freezer b Add the Ligase to the reaction mix at a 1 40 dilution immediately before addition to the sample Ensure that the Ligation Ligase reaction solution is thoroughly vortexed before addition to the sample 14 Duolink In Situ Brightfield Amplification 5x store at 20 C Thaw at room temperature and vortex before use Dilute 1 5 in high purity water immediately before use Polymerase 10 U l store at 20 C gt The Polymerase should be kept at 20 C at all times Use a freezing block when removing the enzyme from the freezer Add the Polymerase to the reaction mix ata 1 80 dilution immediately before addition to the samples Ensure that the Amplification Polymerase reaction solution is thoroughly vortexed before addition to the sample Detection Brightfield 5x store at 20 C gt Thaw at room temperature and vortex before use Dilute 1 5 in high purity water immediately before use and vortex the solution before addition to the sample Substrate A D store at 4
11. 60 min at room temperature 8 SUBSTRATE SOLUTION a Dilute the Substrate A 1 70 B 1 100 C 1 100 and D 1 50 reagents in high purity water E g for a 40 ul reaction add 0 6 ul Substrate A 0 4 ul Substrate B 0 4 ul Substrate C 0 8 ul Substrate D in 378 ul high purity water b Tap off the Detection Brightfield solution from the slides c Wash the slides in 1x Wash Buffer A for 2 x 5 min under gentle agitation d Add the Substrate solution to each sample e Incubate the slides for 5 to 10 min at room temperature 9 NUCLEAR STAIN a Wash the slides in high purity water for 2 x 2 min under gentle agitation b Add the Nuclear Stain to each sample one drop equals 40 yl Note The Nuclear Stain is supplied in working concentration no dilution is required c Incubate the slides for 2 min at room temperature d Wash the slides under running tap water for 10 min 10 DEHYDRATION a 9696 EtOH for 2 x 2 min b 99 7 EtOH for 2 x 2 min c Xylene for 10 min d Move slide to fresh Xylene 11 PREPARATION FOR IMAGING Use a minimal volume of non aqueous mounting medium e g Duolink In Situ Brightfield Mounting Medium and apply a cover slip on top of your sample Preferably add the mounting medium to the cover slip and then gently slip over the sample slide to ensure no air bubbles get caught under the cover slip Work in a well ventilated area e g fume hood and let the slides
12. C gt Thaw at room temperature and vortex before use Dilute in high purity water immediately before use and vortex the solution before addition to the sample Hydrogen peroxide store at 4 C Drop bottle ready for use Nuclear stain store at 4 C gt Drop bottle ready for use 6 2 DUOLINK IN SITU WASH BUFFER The washing steps should be performed in a staining jar with a minimum volume of 70 ml on a shaker cradle with gentle orbital shaking Washing after the primary antibodies should be performed in Duolink In Situ Wash Buffer A or in the wash buffer optimal for your primary antibodies In all subseguent wash steps ensure to use Duolink In Situ Wash Buffer according to protocol Wash Buffer A store at room temperature To prepare a 1x buffer dissolve the content of one pouch in high purity water to a final Store pouches at room temperature 1x solutions may be kept at room temperature for short time storage one week or less For long time storage store at 4 C Bring the Alternatively make your own Duolink In Situ Wash Buffer A according to appendix A gt volume of 1000 ml b solutions to room temperature before use b 6 3 SAMPLES Before you start the Duolink In Situ protocol make sure that your sample on the glass slide is sufficiently pre treated with respect to fixation retrieval and permeabilization see section 5 2 Your reaction area must be delimited with e g a grease pen or silicon mask
13. DuolinkelnSitu BIOSCIENCE The protocols in this manual are compatible with all Duolink In Situ PLA probes and Duolink In Situ Detection Reagents Brightfield Art no 92012 Table of content 1 2 9 INTRODUCTION sms satama a eene Ste yes teet PRINCIPLE OF THE ASSAY ette riter tede APPLICATIONS acre pasa 3 1 Detect and quantify protein interactions 3 2 Detect and quantify protein modification 3 3 Detect and quantify protein expression REAGENTS AND EQUIPMENT 41 Duolink II reagents 4 2 Reagents to be supplied by the user 43 Equipment needed 44 Duolink ImageTool software for image analysis ASSAY CONSIDERATIONS 5 1 Choice of primary antibodies 5 2 Primary antibody optimization 5 3 Sample type 5 4 Pre treatment 5 5 Staining and mounting JM REAGENT PREPARATION 6 1 Duolink Il reagents 6 2 Duolink II Wash Buffer 6 3 Samples 6 4 Reaction volume ASSAY PROTOCOL 7 1 PLA probe protocol CUSTOM solutions 7 2 PLA probe protocol Duolink II Solutions 7 3 Proximity Ligation protocol RESULTS 8 1 Typical results 8 2 Image acquiSitigI ouo urere toe aa regi ges 83 Duolink ImageTool Image analysis s APPENDIX A DUOLINK II WASH BUFFER FORMULA sett REFERENCES Duolink Il Wash Buffer A sese Duolink In Sltu
14. Reagents Brightfield is recognized as a red brown spot see Figure 10 in a brightfield microscope Brightfield microscopy images are usually taken in one focal plane An individual PLA signal is sub micrometer in size and can therefore be above or below the current focus To reduce the number of PLA signals that are out of focus use an objective with high numerical aperture A true PLA signal can be scanned through by changing the focus making it appear and disappear This does not apply to coalesced signals which may occur for highly expressed proteins see Figure 11 To detect all signals it might be necessary to obtain images throughout the entire thickness of the sample However you may acquire images in one plane as long as all images to be compared are acquired in a similar position within the sample As with all images that are to be analysed visually or by software it is important to keep all settings constant during an experiment with regards to light intensity exposure time and grey filters used etc The light intensity in combination with the exposure time should be set to give the correct over under exposure balance Other functions that can influence an analysis are to use the right shading correcting and to do a white balance Duolink In Sltu Brightfield 21 8 3 DUOLINK IMAGETOOL IMAGE ANALYSIS To analyze the results you may use Duolink ImageTool to obtain objective quantification of PLA signals However this software
15. Sci USA 101 8420 24 2004 Fredriksson S Gullberg M Jarvius J Olsson C Pietras K Gustafsdottir SM stman A and Landegren U Protein detection using proximity dependent DNA ligation assays Nat Biotechnol 20 473 77 2002 Duolink In Sltu Brightfield 27 This product is for research use only Not for use in human diagnostic or therapeutic procedures This product includes a license for non commercial use of the Duolink product Commercial users will reguire additional licenses Please contact Olink AB for details There are no warranties expressed or implied which extend beyond this description Olink AB is not liable for property damage personal injury or economic loss caused by this product The following trademarks are owned by Olink AB Olink amp Olink Bioscience Duolink amp and PLAG NovaRED is a trademark of Vector Laboratories Burlingame USA This product is covered by several patents and patent applications including US 6 511 809 US 6 558 928 US 6 8785 15 US 7074 564 US 5 665 539 and related US and foreign patents For use only as licensed by Amersham Biosciences Corp part of GE Healthcare Bio Sciences and Molecular Staging Inc The Phi 29 DNA polymerase may not be re sold or used except in conjunction with the other components of this product See U S Patent Nos 5 854 033 6 124 120 6 143 495 5 001 050 5 198 543 5 576 204 and related U S and foreign patents O 2012 Olink AB All third party trad
16. antibodies matching a set of PLA probes Ethanol and Xylene for rehydration and dehydration of the FFPE tissue samples High purity water sterile filtered MilliQ or similar v v v v Non agueous mounting medium 4 3 EOUIPMENT NEEDED b Brightfield microscope Staining jars b Penor mask for delimitation of reaction area grease pen or silicon mask e g ImmEdge Pen from Vector Laboratories Shaker Humidity chamber moist chamber Freeze block for enzymes Incubator 37 C Pipettes covering the range from 1 ul to 1000 yl v v v v v v Cover slips 4 4 DUOLINK IMAGETOOL SOFTWARE FOR IMAGE ANALYSIS The result from a Duolink In Situ experiment is typically a number of distinct spots PLA signals of sub micrometer size in various locations of the studied cells The Duolink ImageTool may be used to obtain objective quantification of PLA signals however this software is primarily designed for counting PLA signals in images generated from fluorescence microscopy The software automatically detects the nuclei and the cytoplasm size is estimated enabling single cell statistical analysis of expression levels in tissue or cell populations Furthermore regions of interest can be defined a feature of particular relevance when studying tissue samples Raw imaging data can be imported directly from the four major microscope vendors Olympus Leica Nikon and Zeiss The results data can be exported into an Excel sheet for furth
17. compatible with all antigen retrieval methods typically used for IHC including Heat Induced Epitope Retrieval HIER b Enzyme treatment with Proteinase K Pepsin Trypsin etc For HIER it might be necessary to try a few different solutions of various pH Free aldehydes that might cause unspecific binding of your primary antibodies to the sample after HIER can be quenched by washing the samples for 5 min in 2 uM glycine in PBS 5 4 2 QUENCHING OF ENDOGENOUS PEROXIDASE ACTIVITY By incubating your tissue samples in hydrogen peroxide H O the possible endogenous peroxidase activity found in tissue will be suppressed The time recommended in this protocol is optimized on tissues that are FFPE Duolink In Sltu Brightfield 11 12 5 4 3 BLOCKING SOLUTION AND ANTIBODY DILUENT It is essential to use a proper blocking solution and antibody diluent Use the recommendations from the vendor of your primary antibodies if available If you have previously optimized your assay in for example IHC use the same conditions for Duolink In Situ There are two different protocols depending on whether you use your own blocking solution or the Duolink In Situ Blocking Solution PLA probe protocol CUSTOM solutions gt Use this protocol if you use your own blocking and antibody diluent recommended see section 7 1 The same agent used for blocking of the sample should be present also in the antibody diluent preferably together with some deter
18. emarks are the property of their respective owners Olink Bioscience O L 1 N K Dag Hammarskj lds v 52B SE 752 37 Uppsala Sweden BIOSCIENCE www olink com 0656 v 3 0 2012 03 12
19. er b Tap off the primary antibody solution from the slides c Wash the slides in a wash buffer suitable for your primary antibody Washing should be performed in a staining jar with a minimum volume of 70 ml on a shaker cradle with gentle orbital shaking Bring the wash buffer to room temperature before use d Add the diluted PLA probe solution e Incubate the slides in a pre heated humidity chamber for 1 h at 37 C gt gt Proceed to the Detection protocol in section 73 on page 18 16 Duolink In Situ Brightfield 7 2 PLA PROBE PROTOCOL DUOLINK IN SITU SOLUTIONS Follow this protocol if you use the Duolink In Situ Blocking Solution for blocking of your sample and Duolink In Situ Antibody Diluent for dilution of your primary antibodies Before you start your samples should be deposited on glass slides and pre treated with respect to fixation retrieval and or permeabilization Use open droplet reactions without a cover slip and perform all incubations in a humidity chamber Use volumes corresponding to your delimited reaction area see the Reaction Volume Guide Art no 80520 or section 6 4 To avoid dilution of the reagents tap off as much wash buffer as possible without letting the sample dry 1 PEROXIDASE QUENCHING a Add the Hydrogen Peroxide solution to each sample Note Optimization of incubation time may be needed b Incubate at room temperature for 5 min c Wash the slides wit
20. er evaluation Duolink In Situ Brightfield 5 Assay considerations 5 1 CHOICE OF PRIMARY ANTIBODIES The Duolink In Situ reagents are generic reagents using secondary antibodies for detecting presence of analyte specific primary antibodies Your choice of primary antibodies is crucial when setting up the PLA assay The primary antibodies should be of IgG class specific for the target to be detected and preferably affinity purified The primary antibodies could be either polyclonal or monoclonal To maximize your success rate choose antibodies that are IHC and or IF classified and follow the optimization guidelines given in section 5 2 Primary antibodies could also be qualified individually in a Duolink In Situ single recognition experiment section 5 1 1 A before a double recognition assay 5 1 1 PRIMARY ANTIBODIES FOR DETECTION OF PROTEIN EXPRESSION When single protein targets are to be detected you can use either one or two primary antibodies against your target A One primary antibody single recognition e Usage of one primary antibody gives you an easy to set up assay e J with high sensitivity In this case only one antibody needs to bind gt the target under the conditions used fixation retrieval buffer etc Using one primary antibody is only recommended for detection of single target proteins when a good performing specific primary antibody is available If you use an unspecific antibody you will see high backgr
21. gent to reduce background staining as well as in the diluent for the PLA probes Note Ensure that you do not use bulk IgG from the same species as your primary antibodies as blocking reagent as this will cause false signals from the PLA probes PLA probe protocol Duolink In Situ Solutions Use this protocol if you have no previous experience or no recommendation from the antibody vendor regarding blocking and antibody diluents see section 72 Note The Duolink In Situ Antibody Diluent contains salt blocking agent and detergents All to prevent unspecific binding of the antibodies It is important that you first verify the function of your primary antibodies one at the time using Duolink In Situ single recognition or possibly IF or IHC Use proper controls to verify that your individual antibodies do bind the correct target under the conditions used 5 5 STAINING AND MOUNTING 5 5 1 DEVELOPING The Detection Reagent contains an oligonucleotide labeled with horse radish peroxidase HRP Also included is the HRP substrate NovaRED M which together with HRP forms a visible enzyme substrate complex This complex will appear as a red or red brown product in a brightfield microscope Developing time for NovaRED substrate in IHC is usually decided by the investigator and varies between 5 15 min The developing time appears to depend somewhat on the pretreatment of your sample The enzyme based pretreatments produce dots of a larger size
22. h 1x Wash Buffer A for 2 x 5 min under gentle agitation 2 BLOCKING a Add one drop of Blocking Solution per 1 cm2 Ensure to cover the entire reaction area with Blocking Solution b Incubate the slides in a pre heated humidity chamber for 30 min at 37 C 3 PRIMARY ANTIBODIES a Mixand dilute your primary antibody or antibodies to a suitable concentration in Antibody Diluent b Tap off the Blocking Solution from the slides Try to obtain an equal residual volume on each slide as this will affect reproducibility Do not allow the samples to dry before adding the primary antibodies as this will cause background c Add the primary antibody solution to each sample d Incubate in a humidity chamber Use temperature and time optimal for your primary antibodies 4 PLA PROBES a Mixand dilute the two PLA probes 1 5 in Antibody Diluent E g for a 40 ul reaction take 8 ul of PLA probe MINUS stock 8 ul of PLA probe PLUS stock and 24 ul of Antibody Diluent b Tap off the primary antibody solution from the slides c Wash the slides in 1x Wash Buffer A or in a wash buffer suitable for your primary antibody preferably 2 x 5 min Washing should be performed in a staining jar with a minimum volume of 70 ml on a shaker cradle with gentle orbital shaking Bring the wash buffer to room temperature before use d Add the PLA probe solution e Incubate the slides in a pre heated humidity chamber for 1 h at 37 C
23. is primarily designed for counting PLA signals in images generated from fluorescence microscopy By using the software it is possible to obtain either the number of signals and cells per image allowing average measurements or to allocate each individual signal to a specific cell using single cell analysis The nuclei are automatically detected and cytoplasm size estimated enabling single cell statistical analysis of expression levels in tissue or cell populations A result can typically look as shown in Figure 12 Fig 12 Analysis of an image from a brightfield microscope Nuclei are blue the red brown spots are the PLA signals representing the protein target of interest In the analysis PLA signals marked with red circles and nuclei outlined in yellow are quantified at analysis The green outlines represent the user defined cytoplasm size When studying highly expressed proteins the density of PLA signals may be so high that it is impossible to discriminate between individual signals see Figure 11 Duolink ImageTool can not quantify regions of merged signals only separable signals can be counted In this case the data is best analyzed by intensity measurement of the area fraction of the sample with signal using software for traditional image analysis The primary antibodies used in the assay can be titered down to reduce the number and incidence of merged signals 22 Duolink In Situ Brightfield 9 Troubleshooting
24. lution and PLA probes to be in contact with your blocking reagent before addition to the sample Dilute the PLA probes with your own diluent the same as you used for blocking and let this incubate in RT for 20 min before applying it to the slides Not sufficient Ensure sufficient washing and use fresh wash solutions and clean washing of slides jars Drying of sample Ensure good humidity during all incubation steps and never let slides dry out after washes and before addition of reagents Unspecific Titrate primary antibodies IHC or PLA with respect to binding of concentration temperature time and buffer and ensure appropriate primary fixation protocol was used antibodies Unspecific Avoid drying of samples during incubation and extended incubation binding of times at high concentration of PLA probes If you want to use secondary serum as an antibody diluent it is possible to use normal donkey reagents serum together with the anti species PLA probes PLA probes directed against species are all raised in donkey Brown Not sufficient Endogenous peroxidase is still active in the specimen and will thus signals or H O quenching develop the substrate shading in your Too long If the developing will proceed for too long the background will negative developing time increase as a brown shadow over the staining area control The No or insufficient Use a grease pen or alike to encircle reaction area reactio
25. me conditions fixation retrieval buffer etc Fig 8 Protein modifications 5 1 3 PRIMARY ANTIBODIES FOR DETECTION OF PROTEIN INTERACTIONS Duolink In Situ provides an excellent way to detect protein c interactions and or heterodimeric targets This is done using two a primary antibodies each directed against one of the targets of interest The two primary antibodies must be raised in different species and must bind to the target under the same conditions fixation retrieval buffer etc Nom Fig 9 Protein interactions 5 2 PRIMARY ANTIBODY OPTIMIZATION You will save time and increase your success rate if you optimize your primary antibodies to be sure that they work properly in your material The conditions for your primary antibodies should be optimized with respect to Sample fixation Antigen retrieval Blocking solution Antibody diluent Primary antibody concentration v v v v v Sample pre treatment for Duolink In Situ is identical to procedures used for immunohistochemical IHC and or immunofluorescence IF staining If you already have a working assay for IHC or IF use the same pre treatment protocol The primary antibody concentration may need to be titrated and optimized for the Duolink In Situ conditions When using two primary antibodies optimization is best done for one primary antibody at the time using the Duolink In Situ single recognition approach These conditions can then be used as a
26. n delimitation spreads around reaction over a area too large surface Inappropriate Adjust reaction volume according to your reaction area For is unable reaction volume guidance on reaction volumes please refer to section Reagent to cover Preparation or the separate Reaction Volume Guide sample 24 Duolink In Situ Brightfield PROBLEM PROBABLE CAUSE SUGGESTED SOLUTION No nuclear o addition Ensure that the Mayers Hematoxylin will be added and that it is staining or of Mayers prediluted and ready to use red nuclear Hematoxylin staining is observed o maturation of The maturation should be allowed for 10 min in running tap water It Hematoxylin will not work as well to use cleaner water for this step Difficulties Sections of Try to use sections that are 4 um or thinner The size of the Duolink in focusing issue are too In Situ HRP signals are 0 3 0 5 um in diameter It is a challenge signals in hick to get the focus plane containing most of the signals but it will be specimen easier when the section is thin If problems remain please contact us at support olink com or 46 18 444 3970 Duolink In Sltu Brightfield 25 Appendix A Duolink In Situ Wash Buffer formula DUOLINKIN SITU WASH BUFFERA Alternative 1 use Duolink In Situ Wash Buffer A pouches To prepare a 1x buffer dissolve the content of one pouch in high purity water to a final volume of 1000 ml Store pouches at room tempe
27. of Polymerase into account when calculating the amount of water added E g for a 40 ul reaction take 8 ul of the 5x Amplification stock and 31 5 ul of high purity water b Tap off the Ligation Ligase solution from the slides c Wash the slides in 1x Wash Buffer A for 2 x 2 min under gentle agitation Tap off all wash solution after the last washing d Add Duolink In Situ Polymerase to the Amplification solution from step a at a 1 80 dilution and vortex E g for a 40 ul reaction add 0 5 ul of Polymerase to 39 5 ul of Amplification solution e Add the Amplification Polymerase solution to each sample f Incubate the slides in a pre heated humidity chamber for 90 min or 120 min at 37 C Note The amplification time is dependent on the epitope retrieval process in your pre treatment protocol Heat Induced Epitope Retrieved HIER slides should be amplified for 120 min and Enzyme Induced Epitope Retrieved EIER slides for 90 min 18 Duolink In Situ Brightfield 7 HRP LABELED PROBES a Dilute the Detection Brightfield stock 1 5 in high purity water and mix E g for a 40 ul reaction take 8 ul of the 5x Detection stock and 32 ul of high purity water b Tap off the Amplification Polymerase solution from the slides c Wash the slides in 1x Wash Buffer A for 2 x 2 min under gentle agitation d Add the Detection Brightfield solution to each sample e Incubate the slides in a humidity chamber for
28. old Ensure that no excessive amount of wash solution remains on the slide before addition of amplification reagents Keep amplification time and temperature Ensure Polymerase is active i e has been kept at 20 C and that correct dilution of the reagents have been used Prepare fresh dilutions just before use do not allow mix with enzyme to stand for more than 5 minutes before use Inefficient If the signals are weak they can appear to be few as only a fraction detection reaches above detection threshold Ensure that the Detection stock or solution has not been exposed to excessive heating or extended time of light during handling or incubation Ensure that the reagents were correctly diluted Duolink In Sltu Brightfield 23 PROBLEM PROBABLE CAUSE SUGGESTED SOLUTION Large Uneven spread Ensure that entire area of investigation is covered during each step variation of reagents of signals during different over slide steps individual reaction Partial drying of Check before each new step that no region has dried during parts of the area incubation If so use a better humidity chamber during incubation High Incomplete f you use paraffin embedded samples incomplete removal of background deparaffinization paraffin can cause background Use fresh solutions if necessary general and ensure correct times during deparaffinization cause Custom blocking f you have used your own blocking solution allow primary antibody so
29. ound due to the sensitivity of the assay Using only one primary antibody is more efficient than using two primary antibodies when detecting low abundant targets Fig 6 A Single recognition B Two primary antibodies double recognition Usage of two primary antibodies will give you an assay with superior specificity When using two primary antibodies they must be directed against different non competing epitopes on the same target molecule The two primary antibodies must have been raised in different species Also both primary antibodies must bind to the target under the same conditions fixation retrieval buffer etc Fig 7 B Double recognition Duolink In Sltu Brightfield 9 5 1 2 PRIMARY ANTIBODIES FOR DETECTION OF PROTEIN MODIFICATIONS Detection of protein modifications such as phosphorylations f J often suffers from low specificity Our recommended strategy is n f to use two primary antibodies one against the target protein and one against a modification site on the same protein Preferably a specific modification site is targeted but it is also possible to use a generic antibody against a modification site Using two primary antibodies will give you superior specificity for your modification assay compared to using one single primary antibody The two primary antibodies must be raised in different species Also both primary antibodies must bind to the target under the sa
30. rature Expiry date is marked on each individual lot 1x solutions may be kept at room temperature for short time storage one week or less For long time storage store at 4 C Bring the solutions to room temperature before use Alternative 2 make your own Duolink In Situ Wash Buffer A Dissolve 8 8 g NaCl 1 2 g Tris base and 0 5 ml Tween 20 in 800 ml high purity water Adjust pH to 74 using HCI Add high purity water to 1000 ml final concentrations 0 01 M Tris 0 15 M NaCI and 0 0596 Tween 20 Filter the solution through a 0 22 um filter and store at 4 C Bring the solutions to room temperature before use 26 Duolink In Situ Brightfield References Jarvius M Paulsson J Weibrecht I Leuchowius KJ Andersson AC W hlby C Gullberg M Botling J Sj blom T Markova B stman A Landegren U S derberg O In situ detection of phosphorylated platelet derived growth factor receptor p using a generalized proximity ligation method Molecular and Cellular Proteomics 6 1500 1509 2007 S derberg O Gullberg M Jarvius M Ridderstr le K Leuchowius KJ Jarvius J Wester K Hydbring P Bahram F Larsson LG and Landegren U Direct observation of individual endogenous protein complexes in situ by proximity ligation Nat Methods 3 995 1000 2006 Gullberg M Gustafsdottir SM Schallmeiner E Jarvius J Bjarneg rd M Betsholtz C Landegren U and Fredriksson S Cytokine detection by antibody based proximity ligation Proc Natl Acad
31. solution in combination with a xylene based mounting medium Duolink In Situ Brightfield Mounting Medium Art no 80102 5 5 4 MOUNTING The specimen is developed with NovaRED substrate and should subsequently be dehydrated cleared and then mounted with an organic mounting media The red brown staining product can darken with time but this will not affect the staining quality Please observe that you should not use an aqueous mounting medium Duolink In Situ Detection Reagents Brightfield has been evaluated using Duolink In Situ Brightfield Mounting Medium a xylene based mounting medium Please work in a well ventilated area e g fume hood and let the slides dry well before analyzing in the microscope 5 6 CONTROLS To be able to properly evaluate your results it is advisable to include both biological and technical controls Examples of suitable controls are given below 5 6 1 POSITIVE CONTROL Ideally you should include a cell or tissue type that you know contains your target protein and that your primary antibodies should bind to If you are using two primary antibodies the primary antibodies should be known to bind in close proximity This also enables you to verify the Duolink In Situ procedure 5 6 2 NEGATIVE CONTROL Ideally you should include a negative control with a cell line or tissue that does not express one or both of your targets Such a control will give you information on the specificity of your primary antibodies If
32. start for optimizing your assays using both primary antibodies in the same Duolink In Situ assay 10 Duolink In Situ Brightfield When using two primary antibodies it may in some cases be difficult to find conditions compatible with both of the selected primary antibodies Under these circumstances it is advisable to search for other antibodies recognizing alternative epitopes For further advice on immunostaining optimization we recommend the Education Guide from Dako http pri dako com 08002 25may06 ihc guide book pdf 5 3 SAMPLE TYPE Duolink In Situ Detection Reagents Brightfield performs well with Formalin Fixed Paraffin Embedded FFPE tissue sections 5 4 PRE TREATMENT Before you start make sure that your sample on the glass slide is sufficiently pre treated with respect to fixation retrieval and permeabilization It is crucial for the performance of the assay to optimize the conditions for the primary antibodies Also the choice of wash buffer may be dependent on your primary antibodies Use the recommendations from the vendor of your primary antibodies if available 5 4 1 ANTIGEN EPITOPE RETRIEVAL Antigen epitope retrieval is mostly used for FFPE material Formalin fixation is masking the epitopes which then can be un masked by antigen epitope retrieval Different methods for antigen epitope retrieval will influence the performance of your primary antibodies and must be optimized for your application Duolink In Situ is
33. t primary antibodies one directed against the target protein and one against a modification site on the same protein 3 3 DETECT AND QUANTIFY PROTEIN EXPRESSION Detection and quantification of protein expression can be done with two different approaches A To detect and quantify one single protein with high sensitivity use only one primary antibody B To detect and quantify one single protein with high specificity use two different primary antibodies directed against two different epitopes on the same protein Duolink In Situ Brightfield Fig 2 Protein interactions Fig 3 Protein modifications Fig 4 A Single recognition Fig 5 B Double recognition 4 Reagents and eguipment 4 1 DUOLINK IN SITU REAGENTS The Duolink In Situ reagents are generic reagents using secondary antibodies for detecting presence of analyte specific primary antibodies provided by the user To run a Duolink In Situ Brightfield assay you need the following Duolink In Situ components PLA probe MINUS PLA probe PLUS Detection Reagents Wash Buffer A Mounting Medium v v v v v 4 1 1 DUOLINK IN SITU PLA PROBES The choice of PLA probes depends on the species of your primary antibodies and your application see section 3 For a complete list of PLA probes visit www olink com Each Duolink In Situ PLA probe product contains the following gt Blocking Solution For blocking
34. this control gives you background signals in excess of signal obtained from omitting the primary antibody antibodies it means that your primary antibodies under the present conditions are binding to other targets than expected If facing problems with signals in your negative cell line tissue you might need to titrate your primary antibodies or look for more specific antibodies 5 6 3 NEGATIVE CONTROL TECHNICAL By omitting primary antibodies you will get a hint of how the PLA probe background looks like in your system Alternatively if you are using an approach with two primary antibodies you can also choose to omit only one of your primary antibodies If facing problems with background signals with the primary antibodies omitted consult the troubleshooting guide see section 9 Duolink In Sltu Brightfield 13 6 Reagent preparation 6 1 DUOLINK IN SITU REAGENTS Some Duolink In Situ reagents are supplied as concentrated stocks Dilute reguired volumes of the stocks Note Do not store diluted reagents Blocking Solution store at 4 C b Vortex before use b Ready to use blocking solution one drop equals approximately 40 ul Antibody Diluent store at 4 C b Vortex before use b For dilution of primary antibodies and PLA probes PLA probe 5x store at 4 C b Vortex before use b Dilute 1 5 in Antibody Diluent or custom diluent immediately before use and vortex the solution before addition to
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