Immunohistochemical Detection With Quantum Dots
Contents
1. C Bisbenzimide Hoescht 33 258 stock solution prepare Hoescht 33 258 Sigma cat no B 2883 at 2 mg mL stored at 4 C Dilute 1 uL in 10 mL PBS when apply ing to slides 90 v v Glycerol in PBS Fisher Scientific cat no G33 1 Microscope cover glasses various sizes VWR International cat no 48393 Optical fluorescence filters usable excitation filters include 325 25 nm UV 360 40 nm DAPI 470 40 nm FITC or 545 30 nm rhodamine Usable emission filters include 400 nm long pass 450 50 nm DAPI 535 40 nm FITC or 610 75 nm Cy3 Custom filtersets for QDs are available from Chroma Technology Rockingham VT or Omega Optical Brattleboro VT 2 3 Simultaneous Detection of Two Antibodies Raised in Different Species Using QD Conjugated Secondary Antibodies l 2 3 4 Mouse anti MAP2 primary monoclonal antibody Sigma cat no M4403 Rabbit anti GFAP primary antiserum Dako Glostrup Denmark cat no Z334 Quantum dot 565 anti rabbit secondary antibody QD 565 goat F ab 2 anti rabbit IgG conjugate Quantum Dot Corp cat no 1143 1 QD 605 anti mouse secondary antibody QD 605 goat F ab 2 anti mouse IgG conjugate Quantum Dot Corp cat no 1100 1 2 4 Detection of Single Antibody Immunoreactivity by TSA and a QD Conjugated Streptavidin 1 3 Hydrogen peroxide dilute stock 30 hydrogen peroxide Fisher Scientific cat no BP2633 in PBS at time of use Store stock solut2. In this chapter we describe how to use QD conjugates in several immunohisto chemical applications We demonstrate multilabeling QD protocols and how to combine QD conjugates with tyramide signal amplification TSA for exquisitely sensitive immunohistochemical detection Finally we describe how to combine these techniques to accomplish simultaneous triple labeling For completeness we begin with an overview of our tissue section preparation protocol 2 Materials 2 1 Preparation of Tissue for Immunohistochemical Study 1 10X Phosphate buffered saline PBS dissolve 80 g NaCl 2 0 g KCl 14 2 g Na HPO 2 0 g KH PO and 0 1 g NaN in I L distilled H O Adjust pH to 7 2 with NaOH Store 10X solution at room temperature Dilute 10X using distilled H 0 for 1X PBS and store at room temperature 2 4 w v Paraformaldehyde dissolve 4 g paraformaldehyde Electron Microscopy Sciences Fort Washington PA cat no 19200 in PBS heat to 58 60 C and add I N NaOH dropwise until solution is clear Fill to 100 mL volume with PBS and pH to 7 2 Use immediately If solution is heated above 60 C do not use 3 Bouin s fixative 750 mL saturated aqueous picric acid Sigma St Louis MO cat no P 6744 250 mL formaldehyde solution Fisher Scientific Pittsburgh PA cat no F75 1GAL and 50 mL glacial acetic acid Fisher Scientific cat no A58 500 Store at 4 C 4 Tissue freezing medium VWR International West Chester PA cat no 151
3. solution in the PBS during the second wash Cover slip slides in 90 glycerol in PBS see Note 10 View slides with a fluorescence microscope equipped with a 360 40 nm DAPI excitation filter and a 400 nm long pass emission filter see Note 11 see Subheading 3 7 for information on optimal filterset selection 3 3 Simultaneous Detection of Two Antibodies Raised in Different Species Using Two QD Conjugated Secondary Antibodies There are several variations and extensions of the protocol in Subheading 3 2 that will allow the detection of multiple antigens in fixed tissue Multiple primary antibodies can be applied simultaneously to a single tissue section After an overnight incubation period multiple secondary antibodies can then be applied to detect primary antibody binding As long as each primary antibody is raised in a different species this technique retains specificity and is very straightforward An example of double antibody immunoreactivity detection with two QD conjugated secondary antibodies is shown in Fig 2 il Begin immunostaining as described in Subheading 3 2 steps 1 4 2 Dilute multiple primary antibodies directed toward antigens of interest in the same 2 antibody cocktail For example one can make a cocktail containing a 1 uL Mouse derived anti MAP2 antibody b 1 uL Rabbit derived anti GFAP antiserum c 198 uL PBS BB For a 1 200 dilution of both primary antibodies Apply primary antibody c
4. 2 Immunohistochemical Detection With Quantum Dots Rizwan S Akhtar Cecelia B Latham Dario Siniscalco Carlo Fuccio and Kevin A Roth Summary Quantum dot QD conjugates have many immunohistochemical applications The optical excitation emission and photostable properties of QDs offer several advantages over the use of chromogens or organic fluorophores in these applications Here we describe the use of QD conjugates to detect primary antibody binding in fixed tissue sections We also describe the use of QDs in simultaneous and sequential multilabeling procedures and in combination with enzyme based signal amplification techniques QD conjugates expand the arsenal of the immunohistochemist and increase experimental flexibility in many applications Key Words Immunostaining detection methods multi labeling tyramide signal amplification fluorophore antibody binding 1 Introduction Detection of biologically relevant molecules in human and animal fixed tissue sections is often accomplished by applying primary antibodies that bind specifically to the antigen of interest followed by application of labeled second ary antibodies Traditionally the secondary antibodies used in immunohisto chemical detection have been directly conjugated to organic fluorophores These labels are easily visualized using a suitably equipped fluorescence micro scope Alternatively the secondary antibodies may not be labeled per se but rather conjuga
5. 48 031 5 Slides Snowcoat X tra Surgipath Medical Industries Inc Richmond IL cat no 15148 031 6 Slide rack staining dish Wheaton 900200 VWR International cat no 25461 003 7 Antigen retrieval container TPX staining jar VWR International cat no 25460 907 Immunohistochemistry and Quantum Dots 13 oo 11 CitriSolv Fisher Scientific cat no 22 143975 Isopropanol Fisher Scientific cat no HC 500 1GAL Citrate antigen retrieval buffer working solution 10 mM citrate buffer pH 6 0 consists of 9 mL 0 1 M citric acid monohydrate solution Sigma cat no C 7129 41 mL 0 1 M sodium citrate solution Sigma cat no S 4641 and 450 mL dis tilled H O for a total of 500 mL Store all solutions at 4 C Steamer Farberware FRA500 domestic rice cooker or equivalent 2 2 Detection of Single Antibody Immunoreactivity With QD Conjugated Secondary Antibody 1 2 D Plastic slide folder VWR International cat no 48443 850 Pap pen Research Products International Mount Prospect IL cat no 195504 was used in this study Quantum Dot Corporation recommends the use of the ImmEdge Hydrophobic Barrier Pen Vector Labs Burlingame CA cat no H 4000 PBS blocking buffer BB dissolve 1 0 g bovine serum albumin Fisher Scientific cat no BP1600 100 0 2 g nonfat powdered skim milk Carnation Nestle Glendale CA and 0 3 mL Triton X 100 Sigma cat no T 8787 in 100 mL 1X PBS Store at 4
6. 5 conjugated goat derived anti rabbit antibody c 98 uL of QD buffer For a 1 100 dilution of both secondary antibodies 6 Apply secondary antibody cocktail to each slide and incubate for h at room temperature 7 Wash the slides three times in PBS for 5 min each If nuclear counterstaining is desired dilute stock bisbenzimide Hoescht 33 258 solution in the PBS during the second wash 8 Cover slip slides in 90 glycerol in PBS see Note 10 9 View slides with a fluorescence microscope equipped with a 360 40 nm excita tion filter and a 400 nm long pass emission filter which will display both labeled colors Alternatively use a narrow band filter set for each of the QDs used and view the signals individually see Subheading 3 7 for information on optimal filter set selection 3 4 Detection of Single Antibody Immunoreactivity by TSA and QD Conjugated Streptavidin QD streptavidin conjugates can be successfully incorporated in enzyme based signal amplification techniques including TSA 4 Enzyme based signal amplification in immunohistochemical detection methods effectively increases the number of fluorescent molecules at antigenic sites of interest 1 Although several enzyme based signal amplification methods are available TSA is the most versatile TSA can greatly increase the sensitivity of immunohistochemi cal detection thereby aiding the detection of antigens that are expressed at low levels 8 9 Furthermore signa
7. 70 40 nm excitation filter and a 535 40 nm emission filter C Hoescht 33 258 counterstaining of nuclei is imaged with a 360 40 nm exci tation filter and a 450 50 nm emission filter D Calbindin GFAP and Hoescht 33 258 counterstaining is simultaneously visualized with a 325 25 nm excitation filter and a 400LP emission filter Scale bar 40 um 22 Akhtar et al antibody and only indicate binding of the second primary antibody QDs can easily be integrated into this type of protocol as outlined next In Fig 4 TSA using QD conjugated streptavidin is combined with QD secondary antibody to perform double labeling where the two primary antibodies used are raised in the same species 1 Prepare slides as described in Subheading 3 1 see Note 5 2 Begin immunostaining as described in Subheading 3 4 steps 2 4 Next com plete Subheading 3 2 steps 2 4 3 Prepare the first primary antibody diluted in PBS BB For example we will use rabbit derived anti calbindin antiserum at a 1 10 000 dilution At this dilution calbindin antiserum binding will not be detected by QD conjugated secondary antibody which requires a 1 100 dilution Apply primary antibody to each slide and incubate overnight at 4 C Do not apply the second primary antibody at this time 4 Wash the slides three times in PBS for 5 min each 5 Dilute HRP conjugated secondary antibody in PBS BB Apply 50 200 uL of diluted HRP conjugated secondary antibody to each s
8. antibody but do receive all subsequent detection reagents These negative control slides will illustrate any background or false positive signals generated by the detection method that is independent of pri mary antibody binding Some tissue has significant autofluorescent material that will be evident in these negative control slides In addition we have found that if used at inappropriately high concentrations QD reagents may nonspecifically label neurons 4 This nonspecific labeling will also be evident in negative con trol slides and will allow for better interpretation of truly labeled slides Nonimmune serum controls or preabsorbed primary antibody controls are also useful for interpreting immunohistochemical results Immunohistochemistry and Quantum Dots 27 6 10 11 Pap pens are very useful to create a boundary on a slide to prevent the lateral movement of solution from capillary action This way a smaller volume of antibody reagent can be used to cover tissue completely However Pap pen may interfere with QD signals and photostability 7 and we recommend removing Pap pen prior to the addition of QD reagents Removal is accomplished by using a damp paper towel or a fingernail Pap pen is not absolutely required and a grease pencil scoring with a diamond tip or an aerosolized hydrocarbon coating around the tissue can alterna tively be used with similar results Quantum Dot Corporation suggests the use of the ImmEdge Pap p
9. c day 13 mouse telencephalon A Musashi a marker of neural precursor cells in the ventricular zone is immunostained with a rabbit polyclonal antiserum subsequently detected with TSA and QD 525 conjugated streptavidin and imaged with a 470 40 nm excitation filter and a 535 40 nm emission filter B TuJ1 a marker of immature neurons in the intermediate zone is immunostained with a mouse monoclonal antibody subsequently detected with QD 605 conjugated goat anti mouse secondary antibody and imaged with a 545 30 nm excitation filter and a 610 75 nm emis sion filter C Hoescht 33 258 counterstaining of nuclei is imaged with a 360 40 nm excitation filter and a 450 50 nm emission filter D Musashi TuJ1 and Hoescht 33 258 counterstaining is simultaneously visualized with a 325 25 nm excitation filter and a 400LP emission filter Scale bar 40 um 20 Akhtar et al 3 Wash slides in running tap water for 5 min Wash sections in PBS for 5 min at room temperature 5 For primary antibody application follow Subheading 3 2 steps 2 7 Note that when using TSA the optimal primary antibody concentration is often dramatically reduced compared with non TSA immunohistochemistry detection techniques 6 Dilute HRP conjugated secondary antibody in PBS BB Apply 50 200 uL of diluted HRP conjugated secondary antibody to each slide and incubate for h at room temperature 7 Wash the slides three times in PBS for 5 min each 8 Dilut
10. ceed Akhtar et al Immunohistochemistry and Quantum Dots 25 400 nm can be used We have had good results using a 325 25 nm or 360 40 nm excitation filter with QDs Using a 460 nm short pass excitation filter would pre clude the simultaneous visualization of QDs and fluorescent dyes such as DAPI or Hoescht 33 258 3 Conventional narrow band filters for fluorophores such as Cy3 or FITC can be used to visualize certain QDs For example a Cy3 filter set containing a 545 30 nm excitation filter a 570 nm long pass dichroic beamsplitter and a 610 75 nm emission filter would adequately allow the visualization of QD 605 The 610 75 nm emission filter while not centered on the 605 nm wavelength would allow the passage of emitted light at 605 nm because of its relatively wide bandwidth 75 nm Furthermore a FITC filter set containing a 470 40 nm ex citation filter a 500 nm long pass dichroic beamsplitter and a 525 40 nm emis sion filter would transmit fluorescence from both QD 525 and 565 Therefore an investment in custom filter sets for QD immunohistochemistry is not absolutely required However specific visualization of QD 565 would require a custom fil terset because that wavelength does not correspond to any other commonly used fluorophore The custom filtersets are optimized for detection of QDs and can be expected to produce the best signal to noise ratio 4 If the simultaneous visualization of several QDs is desire
11. d a long pass emission filter can be used For example QD 525 565 and 605 can be simultaneously visualized with a 470 40 nm excitation filter a 510 nm long pass dichroic beamsplitter and a 520 nm long pass emission filter In this way all three QDs can be seen at the same time and currently our laboratory uses a similar filter for QD visualization If a fourth fluorescent dye is to be visualized such as DAPI or Hoescht 33 258 one can use a 325 35 nm excitation filter a 370 nm long pass dichroic beamsplitter and a 400 nm long pass emission filter When using long pass emission filters there may be a decrease in the signal to noise ratio because Fig 5 Sequential detection of three antibodies with tyramide signal amplification TSA and quantum dot QD conjugated streptavidin and with two QD conjugated secondary antibodies A E Immunostained section of adult mouse brain A NeuN a marker of mature neurons is immunostained with a mouse monoclonal antibody sub sequently detected with TSA and QD 525 conjugated streptavidin and imaged with a 470 40 nm excitation filter and a 535 40 nm emission filter B Glial fibrillary associated protein GFAP is immunostained with a rabbit polyclonal antiserum subse quently detected with QD 565 conjugated donkey anti rabbit secondary antibody and imaged with a 470 40 nm excitation filter and a 535 40 nm emission filter C Microtubule associated protein 2 MAP2 is immunostained wit
12. e biotinylated tyramide in amplification buffer as recommended by the manu facturer Apply biotinylated tyramide to each slide 9 Gently rotate slides in the humidified chamber on an elliptical rotator 25 rpm for 3 10 min at room temperature 10 Wash the slides three times in PBS for 5 min each Remove Pap pen from slides see Note 6 11 Dilute QD streptavidin conjugate in QD buffer at a 1 400 dilution and incubate for 30 min at room temperature prior to use 12 Apply 50 200 uL of diluted QD streptavidin conjugate to each slide and incubate for 30 min at room temperature 13 Wash the slides three times in PBS for 5 min each If nuclear counterstaining is desired dilute stock bisbenzimide Hoescht 33 258 solution in the PBS during the second wash 14 Cover slip slides in 90 glycerol in PBS 15 View slides with a fluorescence microscope equipped with a 360 40 nm DAPI excitation filter and a 400 nm long pass emission filter see Note 11 see Subheading 3 7 for information on optimal filter set selection gt A 3 5 Sequential Detection of Multiple Antibody Immunoreactivity by TSA and QD Conjugated Streptavidin and QD Conjugated Secondary Antibodies The protocol described in Subheading 3 3 can only be used if the primary antibodies employed are raised in different species If the antibodies are raised in the same species e g the detection of two mouse derived primary anti bodies special techniques can be used to reta
13. en from Vector Labs to minimize interference with signal intensity To remove antibody or reagent solutions from a slide firmly tap the slide on its side onto the lab bench Then use a folded dry paper towel to dab over the Pap pen boundary There should be no wetness over the boundary Otherwise solution that is added within the boundary will easily pass to the other side via capillary action This process must be done quickly and carefully to ensure the tis sue section does not dry or become scratched or damaged While slides are in the humidified chamber slide folder ensure that the folder remains as horizontal as possible Be sure that any refrigerator shelves that are used to house slides overnight are level so that slides remain adequately wet Also when removing slides from the chamber for washing the underside of the slide folder leaves should be inspected and washed if any residual antibody or reagent is present The slide folder can then be rinsed and reused for the next step of immunostaining When washing slides it is important to use large volumes of fresh PBS One can also agitate slides at 100 rpm on a rotator for better washing Slides that are cover slipped with 90 glycerol in PBS may have undesirable air bubbles beneath the cover slip To overcome this problem the cover slip should be gently removed and reapplied until the air bubbles are either eliminated or are not over any pieces of tissue QD fluorescence
14. h a mouse monoclonal antibody subsequently detected with QD 605 conjugated goat anti mouse secondary antibody and imaged with a 545 30 nm excitation filter and a 610 75 nm emission filter D Hoescht 33 258 counterstaining of nuclei is imaged with a 360 40 nm exci tation filter and a 450 50 nm emission filter E Merged image of NeuN GFAP MAP2 and Hoescht 33 258 counterstaining Scale bar 40 um 26 Akhtar et al of the additional wavelengths of light transmitted thereby leading to increased visible autofluorescence 4 Notes 1 The protocols outlined here are robust in our laboratory and should be used as starting points for the development of protocols in other laboratories There are a large number of potential fixatives and we recommend that several fixatives be tried during protocol optimization The particular fixative used is selected based on the tissue to be immunostained the requirement for perfusion the stability or availability of the antigen to be detected and the characteristics of the antibody to be used Furthermore there are also many methods of performing antigen retrieval and we recommend that several be tried for each antigen and tissue Washing slides can best be accomplished by placing slides in a suitable glass slide rack that will fit inside a rectangular staining dish and transferring the entire rack from one wash solution to another Transferring individual slides between various solutions fo
15. ime and care should be taken to avoid drying of the tissue 4 To inhibit nonspecific antibody binding incubate the slides in PBS BB blocking buffer for 30 min at room temperature As mentioned in step 3 excess PBS BB can be added to individual slides one at a time Alternatively slides can be processed in bulk by placing the slides in a slide rack and immersing the rack in a staining dish containing PBS BB 5 Prepare primary antibody diluted in PBS BB Remove the PBS BB from the slide see Note 7 and apply primary antibody When applying any antibody or reagent to slides use a sufficient volume to cover the tissue typically 50 200 uL 16 10 11 Akhtar et al Incubate the slides in primary antibody for 24 h at 4 C see Note 8 Incubation can be shortened to 12 h at 4 C or to 60 90 min at room temperature In general longer antibody incubations at lower temperatures result in better signal to noise ratios in immunohistochemical detection Wash the slides three times in PBS for 5 min each see Note 9 Dilute QD conjugated secondary antibody in PBS BB at a 1 100 dilution Because the Pap pen may interfere with QD signals 7 remove the Pap pen from slides before applying QD conjugates see Note 6 Apply secondary antibody and incu bate slides in a humidified chamber for 1 h at room temperature Wash the slides three times in PBS for 5 min each If nuclear counterstaining is desired dilute stock bisbenzimide Hoescht 33 258
16. in specificity while using organic fluorophores or QDs 11 Here we will describe a technique called dilutional neglect using TSA and multiple QDs The first primary antibody is detected using TSA and QD conjugated streptavidin and the second primary antibody is detected by QD conjugated secondary antibody Because TSA significantly increases sensitivity lower concentrations of primary antibody are required for successful immunostaining As long as the first antibody that is to be detected with TSA and QD conjugated streptavidin is used at a lower concentration than that required for detection by QD conjugated secondary antibodies the detec tion method for the second primary antibody will neglect the first primary Immunohistochemistry and Quantum Dots 21 Fig 4 Sequential detection of two antibodies raised in the same species with tyra mide signal amplification TSA and quantum dot QD conjugated streptavidin and with QD conjugated secondary antibody A D Immunostained section of postnatal day 7 mouse cerebellum A Calbindin is immunostained with a rabbit polyclonal anti serum subsequently detected with TSA and QD 605 conjugated streptavidin and imaged with a 545 30 nm excitation filter and a 610 75 nm emission filter B Glial fibrillary associated protein GFAP is immunostained with a rabbit polyclonal antiserum subsequently detected with QD 565 conjugated goat anti rabbit secondary antibody and imaged with a 4
17. ion at 4 C 14 Akhtar et al HRP conjugated secondary antibody peroxidase AffiniPure donkey anti rabbit IgG cat no 711 035 152 and peroxidase AffiniPure donkey anti mouse IgG cat no 715 035 151 both from Jackson ImmunoResearch Laboratories West Grove PA 3 TSA Biotin System Perkin Elmer Life Sciences Products Boston MA cat no NEL700 4 QD 525 streptavidin conjugate Quantum Dot Corp cat no 1014 1 5 QD 605 streptavidin conjugate Quantum Dot Corp cat no 1010 1 3 Methods 3 1 Preparation of Tissue for Immunohistochemical Study 1 Sample tissue should be harvested and immediately fixed in a sufficient volume of fixative such as 4 paraformaldehyde Bouin s fixative or methanol see Note 1 In general overnight immersion fixation at 4 C is satisfactory however for some antigens perfusion fixation and or special fixation procedures may be required for optimal immunohistochemical detection Sample tissue can be prepared as either paraffin embedded or frozen sections Paraffin embedded tissue sections require deparaffinization prior to use Frozen sections should be allowed to warm to room temperature approx 10 min at room temperature prior to further processing Go to step 6 for frozen slides For paraffin sections deparaffinize sections by washing slides twice in CitriSolv first for 10 min and second for 5 min see Notes 2 4 Then rehydrate sections by three washes with isopropanol fo
18. l amplification can increase the ability to detect antigens within an autofluorescent or otherwise noisy sample TSA is based on HRP catalyzed deposition of tyramide conjugated molecules at sites of anti body binding 8 10 11 One can either deposit fluorophore conjugated tyra mide direct TSA or biotinylated tyramide with subsequent detection with a fluorophore labeled streptavidin indirect TSA Next we outline a protocol in which HRP conjugated secondary antibody catalyzes the deposition of biotiny lated tyramide at sites of primary antibody binding Deposited biotin is then detected with QD conjugated streptavidin Figure 3 depicts binding of two pri mary antibodies raised in different species where one primary antibody is detected by TSA and QD conjugated streptavidin and the other primary anti body is detected by QD conjugated secondary antibody 1 Follow Subheading 3 1 to prepare slides see Note 5 2 Incubate sections in 3 hydrogen peroxide in PBS for 5 min at room temperature Hydrogen peroxide will help destroy endogenous peroxidase activity in the tissue that could otherwise lead to false positive signals during the enzyme amplification Immunohistochemistry and Quantum Dots 19 Fig 3 Sequential detection of two antibodies raised in different species with tyra mide signal amplification TSA and quantum dot QD conjugated streptavidin and with QD conjugated secondary antibody A D Immunostained section of embryoni
19. lide and incubate for h at room temperature 6 Wash the slides three times in PBS for 5 min each 7 Dilute biotinylated tyramide in amplification buffer as recommended by the manu facturer Apply biotinylated tyramide to each slide 8 Gently rotate slides in the humidified chamber on an elliptical rotator 25 rpm for 10 min at room temperature 9 Wash the slides three times in PBS for 5 min each 10 Repeat Subheading 3 2 steps 2 4 11 Prepare the second primary antibody diluted in PBS BB In our example we will use rabbit derived anti GFAP antiserum at a 1 100 dilution Apply primary anti body to each slide and incubate overnight at 4 C 12 Wash the slides three times in PBS for 5 min each 13 Prepare QD conjugated secondary antibody and QD streptavidin conjugate in a reagent cocktail at their respective concentrations Be sure the QDs used emit flu orescence at different wavelengths For example the following will detect the primary antibodies used in steps 3 and 11 a 1 uL of QD 605 streptavidin conjugate b 4 uL of QD 565 conjugated goat derived anti rabbit antibody c 395 uL of QD buffer For a 1 400 dilution of QD 605 conjugated streptavidin and a 1 100 dilution of QD 565 conjugated goat derived anti rabbit antibody 14 Apply reagent cocktail to each slide and incubate for 1 h at room temperature 15 Wash the slides three times in PBS for 5 min each If nuclear counterstaining is desired dilute stock bisbenzi
20. may require a brief period of illumination prior to viewing to maximize their signal to noise ratio The immunostained slide should be placed in the light path of the microscope and for 2 4 min for the specific fluorescence to become optimally apparent Acknowledgments We would like to thank all the members of the Roth laboratory for their insights and assistance These protocols were developed in part by support from NIH grants NS35107 and NS41962 RSA received support from the UAB Medical Scientist Training Program NIH grant 08361 References I Roth K A and Baskin D G 2005 Enzyme based fluorescence amplification for immunohistochemistry and in situ hybridization In Molecular Morphology in Human Tissues Techniques and Applications Tubbs R R and Hacker G W eds CRC Press Washington D C pp 65 80 28 10 11 Akhtar et al Bruchez M Jr Moronne M Gin P Weiss S and Alivisatos A P 1998 Semiconductor nanocrystals as fluorescent biological labels Science 281 2013 2016 Wu X Liu H Liu J et al 2003 Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots Nat Biotechnol 21 41 46 Ness J M Akhtar R S Latham C B and Roth K A 2003 Combined tyramide signal amplification and quantum dots for sensitive and photostable immuno fluorescence detection J Histochem Cytochem 51 981 987 Watso
21. mical Detection To successfully view QDs in immunohistochemical applications the appro priate filter sets must be used Investigators may misinterpret their results if an incorrect or suboptimal filter set is used to view the staining General red green blue emission filters may be adequate to passage the narrow emission wavelengths of QDs It is recommended to use custom filter sets for QD view ing but purchasing and maintaining these filters in addition to standard filter sets may not be cost effective Based on our observations we discuss next several aspects of filter set selection for immunohistochemical applications 1 Quantum Dot Corporation recommends a 460 nm short pass excitation filter a 475 nm dichroic beamsplitter and a narrow band pass filter centered at the wave length corresponding to the QD to be viewed For example a QD 605 conjugated secondary antibody should be viewed with a 605 20 nm narrow band pass emis sion filter Custom filtersets that include these filters are currently available from Chroma Technology or Omega Optical 2 QDs are optimally excited at wavelengths below 460 nm and the use of a short pass 460 nm filter will provide maximal excitation No currently available QDs emit light at or below 460 nm However several other dyes such as DAPI and Hoescht 33 258 emit fluorescence at or around 460 nm If it is desired to use these dyes concurrently with QDs an ultraviolet excitation filter that does not ex
22. mide Hoescht 33 258 solution in the PBS during the second wash 16 Cover slip slides in 90 glycerol in PBS see Note 10 Immunohistochemistry and Quantum Dots 23 17 View slides with a fluorescence microscope equipped with a 360 40 nm excita tion filter and a 400 nm long pass emission filter which will display both labeled colors Alternatively use a narrow band filter set for each of the QDs used and view the signals individually see Subheading 3 7 for information on optimal filter set selection 3 6 Triple Labeling Using QD Conjugated Streptavidin and Multiple QD Conjugated Secondary Antibodies In practice several QD conjugated secondary antibodies may be used to simultaneously and specifically detect multiple primary antibody binding in a given tissue sample provided that all primary antibodies are raised in different species Therefore the protocols in Subheadings 3 3 and 3 5 can be combined to achieve triple labeling using QDs Several primary antibodies which will be detected using different QD conjugated secondary antibodies should be pre pared as an antibody cocktail and applied to slides If enzyme based signal amplification is desired it may be performed for one antibody before the use of an antibody cocktail An example of triple primary antibody immuno reactivity detection with a combination of the techniques described here is depicted in Fig 5 3 7 Selecting Filter Sets for Use With QDs and Immunohistoche
23. n A Wu X and Bruchez M 2003 Lighting up cells with quantum dots BioTechniques 34 296 303 Jaiswal J K Mattoussi H Mauro J M and Simon S M 2003 Long term multiple color imaging of live cells using quantum dot bioconjugates Nat Biotechnol 21 47 51 Qdot Streptavidin Conjugates User Manual 2005 Molecular Probes Inc Eugene OR pp 1 14 http probes invitrogen com media pis mp19000 pdf Bobrow M N Litt G J Shaughnessy K J Mayer P C and Conlon J 1992 The use of catalyzed reporter deposition as a means of signal amplification in a variety of formats J Immunol Methods 150 145 149 van Gijlswijk R P Zijlmans H J Wiegant J et al 1997 Fluorochrome labeled tyramides use in immunocytochemistry and fluorescence in situ hybridiza tion J Histochem Cytochem 45 375 382 Bobrow M N Harris T D Shaughnessy K J and Litt G J 1989 Catalyzed reporter deposition a novel method of signal amplification Application to immunoassays J Immunol Methods 125 279 285 Shindler K S and Roth K A 1996 Double immunofluorescent staining using two unconjugated primary antisera raised in the same species J Histochem Cytochem 44 1331 1335
24. ocktail to each slide and incubate overnight at 4 C Wash the slides three times in PBS for 5 min each Remove Pap pen from slides see Note 6 Prepare a single solution of QD conjugated secondary antibodies diluted in QD buffer at their respective optimal concentrations For example one can make the following secondary antibody cocktail to detect the primary antibodies used in step 2 Immunohistochemistry and Quantum Dots 17 Fig 2 Simultaneous detection of two antibodies raised in different species with two quantum dot QD conjugated secondary antibodies A D Immunostained section of postnatal day 7 mouse brainstem A Microtubule associated protein 2 is immunostained with a mouse monoclonal antibody subsequently detected with QD 605 conjugated goat anti mouse secondary antibody and imaged with a 545 30 nm excitation filter and a 610 75 nm emission filter B Glial fibrillary associated pro tein GFAP is immunostained with a rabbit polyclonal antiserum subsequently detected with QD 565 conjugated goat anti rabbit secondary antibody and imaged with a 470 40 nm excitation filter and a 535 40 nm emission filter C Hoescht 33 258 counterstaining of nuclei is imaged with a 360 40 nm excitation filter and a 450 50 nm emission filter D Merged image of MAP2 GFAP and Hoescht 33 258 counter staining Scale bar 40 um 18 Akhtar et al a 1 uL of QD 605 conjugated goat derived anti mouse antibody b 1 uL of QD 56
25. r 5 min each Then wash slides in running tap water for 5 min Finally wash slides in distilled water for 5 min To increase the immunohistochemical detection of some antigenic epitopes in fixed tissue antigen retrieval AR may be required see Note 1 We typically use a citric acid AR method Perform AR by placing slides in an AR container con taining citrate buffer Place the container inside a steamer with an adequate amount of distilled water and turn it on Once steam can be seen exiting the steamer con tinue AR for 20 min Then remove the slide container place it on the lab bench and allow it to cool at room temperature for 20 min Remove slides from AR container and wash in running tap water for 5 min Incubate sections in PBS for 5 min at room temperature Slides are now ready for immunostaining 3 2 Detection of Single Antibody Immunoreactivity With QD Conjugated Secondary Antibody Direct immunofluorescence can be used to detect primary antibody binding in fixed tissue sections Many protocols use organic fluorophore conjugated sec ondary antibodies that recognize species specific epitopes on primary antibodies QD conjugated secondary antibodies can replace organic fluorophore conjugated secondary antibodies in a number of immunohistochemical applications An Immunohistochemistry and Quantum Dots 15 Fig 1 Detection of single antibody immunoreactivity with quantum dot QD conjugated secondary antibody A C Immuno
26. r washing is typically not necessary Also slides can be washed by removing the entire slide rack from the staining dish quickly emptying the dish into the sink replacing the rack in the dish and rapidly pouring a new solution in the dish Care should be taken to minimize the time slides are exposed to air We recommend establishing a deparrafinization station to be used by several investigators in a laboratory Use two staining dishes that contain CitriSolv and three that contain isopropanol Use each dish sequentially to perform depar rafinization and rehydration After several rounds of use replace dish 1 with dish 2 and dish 2 with dish 3 Then clean dish 1 fill with solution and use as dish 2 CitriSolv or 3 Gsopropanol At no time should the tissue on a slide be allowed to dry Dried tissue is perhaps the most frequent cause of suboptimal or uninterpretable staining The tissue must remain moist at all times To ensure that slides remain wet they must be quickly transferred between containers during washes Also the slides should be inspected periodically during incubations with antibody or detection reagents to ensure that solution adequately covers the tissue Even partial or temporary dryness may lead to nonspecific absorption of reagents to the tissue resulting in high background and or nonspecific signals When performing any type of immunohistochemistry control slides should be run in parallel that do not receive primary
27. stained section of postnatal day 7 mouse cerebellum A Calbindin a marker of Purkinje cells in the cerebellum is immunostained with a rabbit polyclonal antiserum subsequently detected with QD 565 conjugated goat anti rabbit secondary antibody and imaged with a 470 40 nm excitation filter and a 535 40 nm emission filter B Hoescht 33 258 counterstain ing of nuclei is imaged with a 360 40 nm excitation filter and a 450 50 nm emis sion filter C Calbindin and Hoescht 33 258 counterstaining is simultaneously visualized with a 325 25 nm excitation filter and a 400LP emission filter Scale bar 40 um example of single antibody immunoreactivity detection with QD conjugated secondary antibody is shown in Fig 1 1 Follow Subheading 3 1 to prepare slides see Note 5 2 Prepare a humidified chamber by cutting a paper towel in half lengthwise and placing it in a plastic slide folder Wet the paper towel with distilled water The paper towel should be damp but not completely saturated with water and should be flat When slides are placed in the folder the underside of the slides should not come into contact with the paper towel 3 Quickly blot excess moisture from around the tissue on each slide with a paper towel and encircle the tissue with a Pap pen or equivalent see Note 6 Place the slides in the humidified chamber and immediately add PBS BB on top of the tissue within the Pap pen boundary One slide should be processed at a t
28. ted to enzymes that catalyze the deposition of chromogenic or fluorescent substrates For example horseradish peroxidase HRP or alkaline phosphatase AP conjugated secondary antibodies can be used to deposit fluorescent or chromogenic substrates at and or near the location of primary antibody binding In this manner enzyme based amplification can greatly increase the sensitivity of immunohistochemical detection 1 From Methods in Molecular Biology vol 374 Quantum Dots Applications in Biology Edited by M P Bruchez and C Z Hotz Humana Press Inc Totowa NJ 11 12 Akhtar et al Quantum dot QD conjugates have recently become commercially available as Qdot Conjugates from Quantum Dot Corporation Hayward CA and can be used in several immunohistochemical applications These reagents offer several advantages over typical fluorophores First many organic fluorophores undergo a rapid and irreversible photobleaching QDs photobleach minimally if at all even with extended periods of viewing 2 5 Second all QDs have a similar excitation spectrum and can be excited in the near 360 nm range This feature coupled with each QD narrow emission spectra facilitates their use in simultaneous multilabeling techniques 2 6 QD based immunohistochemistry can allow multilabeling visualization without the need for computer image overlay or multiple custom filtersets one or both of which is typically required with organic fluorophores
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