AntibodyArrayTM Staining Instruction Manual
Contents
1. Inverted Axiovert 1 and 2 For compatibility with other microscopes please ask With a motorized stage and its control software you can observe one array staining at a time or several array stainings e g staining of different samples with the same staining microarrays together For detailed information on ArrayStage and its control software please contact Hypromatrix Inc There are generic motorized stages from several manufactures which can also be used for ob serving array staining Please contact individual manufacturer for specifications and control software D Protocol I for fixation and permeabilization of cultured cells 1 Grow cells on cover slips until desired state Aspire the medium Fix the cells by 50 Methanol and 50 Acetone cold 20 degree Wash for three times and then put into freezer for 10 to 20 minutes Rinse with PBS for 3 times Blocking in 5 BSA in PBS at 4 degree for 1 hour DN E E Protocol II for fixation and permeabilization of cultured cells 1 Grow cells on a coverslip in a 6 well Wash cells 1x with media without serum and 2x PBS Block the cells 15 in PBS BSA Wash 2x with PBS Fix the cells 20 in 4 paraformaldehyde at room temperature Wash 3x with PBS Permeabilize the cells 10 with 0 2 Triton X 100 Wash 3x with PBS Block the cells 15 in PBS BSA 0 Wash 2x with PBS e ao t sS Hypromatrix Inc 9 www hypromatrix com AntibodyArray Instruction M2. Hypromatrix Inc 11 AntibodyArray Instruction Manual 4 When lifting the tissue on to the slide out of the water bath at no point before or during this process should you touch the flat surface of the slide Hold the slides by the edges or by the front end www hypromatrix com AntibodyArray Instruction Manual V TROUBLESHOOTING If the results you obtained are different from what you have expected use the following guide for troubleshooting For further help please contact Hypromatrix Observations Possible Causes Solutions No signal Cells fell off the support Check cells under microscope to make sure that most cells are still there after the staining Change cell fixa tion conditions or use an additional step to further immobilize cells on the support Use properly treated support to grow cells Improper 2 antibodies Check the 2 antibodies to use correct anti mouse or anti rabbit antibodies Use correct enzyme or fluores cent conjugated antibodies Improper use of Staining AntibodyArrays Make sure that the correct side of the array is con tacted with the cells Follow the instructions Staining is Secondary Antibody is Use high quality enzyme or fluorescent conjugated staining at spots which should be negative proteins other than the supposed antigens in the cells weak not sensitive enough 2nd antibodies Bleaching Use anti
3. bleaching mounting medium and use less bleaching flurophore Insufficient incubation Increase the incubation time time between arrays and cells Too much Blocking is not complete Block the cells overnight background Antibodies contain reac Pre incubate the antibodies with the blocking re tivities to some compo agents Add blocking reagents in wash solution nents in blocking re agents Positive Antibodies recognize Contact Hypromatrix and request some control www hypromatrix com 12 Hypromatrix Inc AntibodyArray Instruction Manual VI REFERENCES Brett D Martin Bruce P Gaber Charles H Patterson and David C Turner 1998 Direct Pro tein Microarray Fabrication Using a Hydrogel Stamper Langmuir 14 15 3971 3975 Ge H UPA 2000 a universal protein array system for quantitative detection of protein protein protein DNA protein RNA and protein ligand interactions Nucleic Acids Res 28 2 e3 Harlow and Lane 1988 Antibodies a laboratory manual Cold Spring Harbor Laboratory Press Lueking A Horn M Eickhoff H Bussow K Lehrach H Walter G 1999 Protein microarrays for gene expression and antibody screening Anal Biochem 270 1 103 111 MacBeath G and Schreiber SL 2000 Printing Proteins as Microarrays for High Throughput Function Determination Science 289 5485 p 1760 1763 Mendoza LG McQuary P Mongan A Gangadharan R Brignac S Eggers M 1999 High throughput microarr
4. 4 Hypromatrix Inc AntibodyArray Instruction Manual II LIST OF COMPONENTS Staining AntibodyArray Store at 4 C Stable for at least 4 weeks HI ADDITIONAL MATERIALS REQUIRED The following materials are needed but not supplied A Cells or tissue sections B Reagents required for cell fixation permeabilization Methanol Acetone Formaldehyde Glutaldehyde C Materials required for performing AntibodyArray staining AntibodyArray staining apparatus Filter papers Filter pads D Solutions 1 Cell fixation permeabilization 50 Aceton 50 Methanol 2 Blocking solution 5 BSA bovine serum albumin in PBS 3 Washing buffer PBS phosphate buffered saline m Reagents required for detection Secondary antibodies fluorophore conjugated goat anti mouse and anti rabbit antibodies 3 Anti bleaching reagent 4 Mounting medium F Equipment required for observing fluorescent immunostaining 1 Fluorescence microscope 2 Motorized stages Hypromatrix Cat HM8020 amp HM8021 3 Stage control Software Hypromatrix Cat HM8010 Hypromatrix Inc 5 www hypromatrix com AntibodyArray Instruction Manual IV METHODS A General considerations 1 Before using staining AntibodyArray tests should be done to get information on the suitability of the cells for AntibodyArray staining Some cells may fall off the support during the staining procedure Cell culture conditions and fixation condi
5. AntibodyArray Instruction Manual I InfroductioD iss cccessacsdssccweccevessnseavadasiens noseseteedueweeandasinsavevesscdecweenssaens 2 m IL List of Component 5 l y p r O m at r xX III Additional Materials Required 5 IV Meth00s asss qsukacassacsauauqascquyyyuqawasaskaqaquwaawpaswuwayaaQqayassqwawuqussws 6 The Proteomics Company A General considerations eee 6 B Protocol for AntibodyArray staining UU 6 C Some considerations for observing and analyzing staining results 7 D Protocol I for fixation and permeabilization of cultured cells 0 6 9 E Protocol II for fixation and permeabilization of cultured cells 9 Antibody Array Staining F Protocol for preparing tissue sections a 10 G Protocol for fixation of frozen sections and cell preps 10 Instru ction Manual H Protocol II for fixation of frozen Sections and cellpreps______ 10 J Steps to take if tissue keeps falling off slides 10 V Troubleshooting scecssscececescccccccececccescscscececscessecesesessesesesesecess 12 VI References E E a 13 VII Appendix I Antibody List by Positions 14 Staining AntibodyArray L ussrsirisiririritiri tninn
6. a motorized stage Because of the large number of antigens needed to be examined for each array staining the staining is best observed under a fluorescence microscope with a motorized stage which allows the movement to each spot automatically However the staining can also be observed without a motorized stage When a motorized stage is not available the AntibodyArray should be aligned with the cells on a coverslip as good as possible The orientation of the staining e g flips rotations during the staining and washing processes should also be recorded This is important for later steps when the staining is observed under a microscope A general procedure to follow when observing a StainingArray without a motorized stage is After staining 1 Mount the coverslip with cells on a slide such that the cells are facing up 2 Align the coverslip with the slide correctly 3 Apply mount medium and overlay another coverslip to cover the cells Try not to move the cells If you did you can correct the movement by inserting a piece of pa per or a razor blade between the slide and the overlaying coverslip to move the cov erslip with the cells Be careful to avoid introducing air bubble 4 Place the slide on the stage of a microscope with the coverslip facing the objective lens Record the orientation 5 Examine the slide under low magnification e g 10X objective first When you move around the field you should see many bright areas ab
7. anual F Protocol for preparing tissue sections 1 Fix the tissue in 10 formalin at 4 C overnight 2 Paraffin embed the fixed tissue 3 Mount tissue sections on slides 5 Clear the paraffin with xylene for ten minutes move slides to a fresh dish of xylene for an additional ten minutes NOTE Perform all xylene washes in a fume hood 6 Rinse the slides twice for 2 minutes in 100 alcohols 18 1 1 100 ethanol 100 methanol 100 isopropanol 7 Rinse the slides twice for 2 minutes in a 95 solution of the 100 alcohols 8 Place slides in an 80 solution of the 100 alcohols for 2 minutes followed by de ionized water for 5 minutes 9 Rinse slides several times with fresh deionized water followed by another five minute wash using fresh water 10 SDS Antigen Retrieval place slides face up in incubation tray and cover each section with 1 SDS in TBS 100mM Tris pH 7 4 138mM NaCl 27mM KCl Incubate for five minutes at room temperature followed by three five minute washes with TBS 11 Blocking immerse slides in a dish containing blocking buffer serum from host spe cies of secondary antibody to be used diluted 1 10 in TBS Incubate at 37 C for one hour G Protocol I for fixation of frozen sections and cell preps 1 Place section or cell prep on slide Air dry 1 hour for cell preps and from 2 hrs to overnight for sections Place in acetone for 5 10 minutes Air dry for 10 minutes Rehydrate by placing 5 minute
8. ay based enzyme linked immunosorbent assay ELISA Biotechniques 27 4 778 80 782 6 788 Wang Y Wu T R Cai S Welte T and Chin Y E 2000 Stat as a component of tumor necrosis factor alpha receptor 1 TRADD signaling complex to inhibit NF kappaB activation Mol Cell Biol 20 13 4505 12 Wang Y 2003 Immunostaining with dissociable antibody microarrays Proteomics In press de Wildt RM Mundy CR Gorick BD Tomlinson IM 2000 Antibody arrays for high throughput screening of antibody antigen interactions Nat Biotechnol 18 9 989 994 Hypromatrix Inc 13 www hypromatrix com AntibodyArray Instruction Manual AntibodyArray Instruction Manual AntibodyArray ining Catalog Number 3 S Nn s A HM8900 AntibodyArray I ining Catalog Number A Sta HM8100 sjods ou J 1 p pUIUUODII 9E POI ut UMOYS H IUQSIH Jo sjods oy 9J0N LH UOJSIH sme l seoog d LH UOJSIH A b dal L sia LH 8U0 sIH L31233 LH uolsiH fail LH uolsiH uluayeo s e18q 71 q e g s 0ds ou r j r p pu uuuroo i ITE POI ut UMOYS JH uolsIH Jo sjods ay 9J0 N aseuly LH UO SIH OLdVZ LAA ABA cAVel aav eges neS EIS e z b qu Z LSOS pews ggwes V oyy dieu O Ldqy 104d qH HES LH uolsiH M Leyo A HS Zdld HS bdid WNOd Law 9d egd zdys gpd os g eddey 4N WAIN LH UO SIH xe dey Yd VW ged L PeEW god g eddey 4N e z b
9. brane in such a manner that when they make contact with cells fixed on another support the antibodies can bind to their respective antigens When the array support is separated from the cells the antibodies will be dissociated from the support and remain bound to the antigens Therefore the method enables the staining of multiple antibodies simultaneously each at a pre determined position AntibodyArray staining technology offers a high throughput method for examining in vivo protein activities It has many applications including 1 Examining protein expressions 2 Revealing protein sub cellular localizations Measuring protein expressions has applications in a variety of fields including biomedical re search disease diagnosis and drug discovery Antibody array staining provides an unique ap proach to reveal protein expression patterns It is useful in comparing the expressions of a large number of proteins between different biological samples see Fig 2 www hypromatrix com 2 Hypromatrix Inc AntibodyArray Instruction Manual ABCDEFGHIJK LMNOPQRST ABCOEFGHIJ KLMNOPQRST ile ae 3 4 6 o 6 ee 7 e a a 5 10 o Fig 2 Comparison of protein expressions between ME180 cells left and A431cells right using antibody arrays with 240 antibodies Alkaline phos phatase labeled secondary antibodies were used and the staining was visualize
10. d by color reaction with BCIP NBT as substrates Knowledge of a protein s sub cellular localization can provide important information about the protein s functional state For example change of a transcriptional factor s location from cytoplasm to nucleus often suggests its activation When observed under a fluorescence mi croscope AntibodyArray staining can reveal sub cellular localizations of many proteins simultaneously Fig 3 Fluorescent staining with staining AntibodyArray An array of 200 rabbit polyclonal antibodies were used and the staining at four positions are shown here as representatives a b transcriptional factor IRF1 c d signaling molecule 14 3 3 B e f cell adhesion protein B catenin g h transcriptional fac tor Ets 1 Low magnification a c e g and i shows the stained cells and sur rounding non stained area and high magnification shows the detailed nuclei lo calization of IRF1 b cytoplasmic staining of 14 3 3 B d and Ets 1 h and membrane staining of B catenin at cell cell contacts d Scale bar in a 300 um Scale bar in b 30 um Simultaneous staining of two proteins double staining is a unique tool for studying two functionally related proteins For example evidence of protein interactions often includes the demonstration that the proteins co localize in the same cellular structure Double staining is also useful when the protein of interest is only expresse
11. d in small number of target cells among a heterogeneous cell population and the protein of interest needs to be observed to gether with a protein marker which is used to denote the target cells Array staining is unique in that it allows the examination of multiple proteins individually as well as simultaneously in the same cell preparation Hypromatrix Inc 3 www hypromatrix com AntibodyArray Instruction Manual Fig 4 Fluorescent double staining with AntibodyArray a Low magnification of A431 cells staining with an array containing rabbit anti YY 1 antibodies left in green mouse anti p130 antibodies middle in red and both YY1 right in green and p130 right in red antibodies at neighboring positions Goat anti rabbit Cy2 labeled secondary antibodies and goat anti mouse Cy3 labeled secon dary antibodies were used b Enlarged view of the double staining of YY1 green and p130 red from a Hypromatrix s Staining Antibody Microrrays are designed for the following applications Revealing a protein s novel functions Understanding molecular mechanisms of a protein s function Dissecting signaling pathways activated by specific stimulations Screening cellular effects of drug candidates Discovering novel diagnostic markers Pa P p i For additional information regarding other applications and custom tailored staining arrays please contact Hypromatrix Inc www hypromatrix com
12. dard flatbed scanner The digitized images can then be analyzed When comparing two stainings the overall intensities must be normalized first This may be accomplished by equalizing the intensities of the corresponding reference spots whose expres sion is identical in control and experimental conditions Then the numerical intensities of the corresponding spots may be calculated and compared Because an antibody will bind both its intended characterized antigen s and some unintended proteins which have similar structure to the immunogen non specific binding there may be some false positive signals To obtain a more quantitative result fluorescence conjugated secondary antibody should be used and array staining should be observed under a fluorescence microscope The problem of non specific binding can be minimized since both the fluorescence intensity and subcellular localization of the antigens can be detected and analyzed Hypromatrix Inc 7 www hypromatrix com AntibodyArray Instruction Manual A general procedure for fluorescent AntibodyArray staining is 1 After array staining place the coverslip in a washing container Remember the orien tation of the coverslip 2 Wash with PBS three times 3 Apply Alexa 488 conjugated goat anti rabbit and goat anti mouse secondary antibod ies to the cells 4 Wash with PBS three times 5 Mount the coverslip on a glass slide and observe under a fluorescence microscope Without
13. err rinne 14 Trial AntibodyArray cccccccecessseeseeecceeececeeeeseeeeeceeeeeeeeeeaneas 14 VIII Appendix II Products from Hypromatrix 16 www hypromatrix com Hypromatrix Inc 1 www hypromatrix com AntibodyArray Instruction Manual I INTRODUCTION Immunochemical staining is a versatile technique for determining both the presence and local ization of an antigen Harlow and Lane 1988 This information is of immense value to bio medical research and clinical medicine Most of the current methods all of which involve incu bating cells with an antibody solution only allow cell staining with one or a few antibodies at a time These methods are not suitable for applications in which the expressions and sub cellular localizations of a large number of different proteins need to be examined a b caer ora Yr a e moO y d Fig 1 Overview of AntibodyArray staining technology a make contact between staining antibody array and adherent cells b incubate arrays and cells to allow antibodies to bind to their antigens c remove array support d detect bound antibodies The AntibodyArray staining method takes advantage of Dissociable Protein Array tech nology which allows the delivery of a large number of proteins to their targets in a position addressable manner In staining AntibodyArray s the antibodies are immobilized on a mem
14. ibody Catalog Number HM2101 Alexa 488 conjugated Goat anti mouse secondary antibody France Germany Catalog Number HM2105 Clinisciences SA BioCat GmbH 147 Avenue Henri Ginoux Im Neuenheimer Feld 581 D Others 92120 Montrouge France D 69120 Heidelberg Germany 1 AntibodyArray staining apparatus www clinisciences com www biocat de Catalog Number HM8000 2 Motorized ArrayStage for upright microscopes Japan United Kingdom Catalog Number HM8020 Funakoshi Co Ltd Cambridge Bioscience 9 7 Hongo 2 Chome 24 25 Signet Court 3 Motorized ArrayStage for inverted microscopes Bunkyo ku Newmarket Road Catalog Number HM8021 Tokyo 113 0033 Cambridge CB5 8LA 4 ArrayStage Control program Japan United Kingdom Catalog Number HM8010 www funakoshi co jp www bioscience co uk Limit liability claims All of the antibodies work under conditions tested by Hypromatrix They may not work in other conditions Hypromatrix may change antibodies without notification Returns will be only accepted with Hypromatrix s authorization Hypromatrix Inc shall have no liability for any damage as the result of improper using of its products www hypromatrix com 16 Hypromatrix Inc
15. ine N wu Lg uuB lui A e uu6 lui geq yzl L Odv q g eddey eudie xS9 7 YO ces s6qo se4 eyde yz 1l eb s13 atal ayz B EE K Z 19H n N zqi ujweutq 3 uo g upo 464 0 uluayeo LH UO SIH Z9PO LAPO ewweb IA ulxeuuy ds segoerd 391 LH UO sIH ZAPO s LH uolsiH N rk a v a www hypromatrix com 15 Hypromatrix Inc 14 Hypromatrix Inc www hypromatrix com AntibodyArray Instruction Manual APPENDIX IL PRODUCTS FROM HYPROMATRIX INC A Staining AntibodyArray s H y p ro m atri X l n C 1 Staining AntibodyArray Catalog Number HM8100 2 Trial Staining AntibodyArray Hypromatrix Inc Telephone 508 797 1700 i Catalo Nube ont 25 Winthrop Street Toll free 800 742 6522 Worcester MA 01604 Fax 508 302 0748 B AntibodyArray s http www hypromatrix com Email contact hypromatrix com 1 Signal Transduction AntibodyArray Catalog Number HM3000 2 Apoptosis AntibodyArray Catalog Number HM4000 3 Cell Cycle AntibodyArray Catalog Number HMS5000 4 Custom AntibodyArray Catalog Number HM6000 C Antibodies 1 Primary antibodies Hypromatrix offers a variety of high quality antibodies For a complete list of antibodies and their specificities please visit our web site at www hypromatrix com 2 Secondary antibodies International Distributors Alexa 488 conjugated Goat anti rabbit secondary ant
16. out 1mm apart separated by dark non stained areas Exam all of the spots quickly first and check the orienta tion of the staining image relative to the staining AntibodyArray 6 Find the reference spots go to the approximate position of a reference spot locate the reference spot by its unique staining pattern e g nuclear staining of Histone H1 Then find additional reference spots 7 Go to the spot of interest by moving the microscope stage You can use the ruler in the microscope eyepiece to measure the moving distance In most cases you should be able to identify the next spot by the fluorescence which is lacking outside the stained areas www hypromatrix com 8 Hypromatrix Inc AntibodyArray Instruction Manual With a motorized stage Array staining is best observed under a fluorescence microscope equipped with a motorized ArrayStage from Hypromatrix which also comes with software specifically written for this purpose ArrayStage from Hypromatrix is compatible with the following manufacturers fluo rescent microscopes Manufacturer Model Nikon Upright E400 E600 E800 E1000 Optiphot I and II Inverted TE200 300 TE2000 TS100 Upright BH BX40 50 60 BX41 51 61 BX45 Inverted LX50 70 IX51 71 81 GX51 71 U nv Upright DMLB DMR DMLM Dialux Laborlux Diaplan Olympus Leica Inverted IDMIR DMIL Zeiss Upright Universal Axioskop 1 and 2 Axioplan 1 and 2 Axiophot 1 and 2
17. s in each of three separate solutions of PBS with 2 BSA to reduce background 6 IMPORTANT Use of methanol or other alcohols at any stage of this process may prevent staining with certain antibodies ARWN H Protocol II for fixation of frozen sections and cell preps 1 Obtain frozen sections 4 or 8 well chamber slides or cover slips containing cultured cells 2 Fix in 4 paraformaldehyde in PBS for 30 min at room temperature or min chamber slides or cover slips in PBS containing 0 05 0 1 Triton X 100 3 For examination of cell surface components methanol or Triton X 100 extraction should not be used 4 After a 30 min incubation in PBS containing 0 05 Twen 20 PBST and 3 BSA I Steps to take if tissue keeps falling off slides 1 Cut no thicker than 4 microns 3 is ideal 2 Dry 2 4 hours at 58 C in oven 4 hours minimum for breast tissue or other fatty tis sue You can also dry overnight at 37 C Finally there are microwave methods of drying tissue but the protocol depends on your microwave and the number of slides Generally a 1000W microwave can dry up to 100 slides in about 3 min utes You should examine each slide to make sure the paraffin has melted uniformly across the tissue section 3 Make sure you use positively charged slides If the tissue adhesion problem coin cides with the use of a newly received shipment of slides you may have a bad lot of slides www hypromatrix com 10 Hypromatrix Inc
18. scope 1 For comparing protein expressions all samples were processed in parallel and the enzymatic reactions can be stopped by washing off substrates with PBS 2 Care should be taken to avoid trapping air between cells and arrays 3 Arrays should be carefully aligned with cells and the alignment should be recorded to avoid any confusion at later steps 4 After arrays contact cells any movement between them should be avoided The in teraction between antibodies and antigens begins in minutes C Some considerations for observing and analyzing staining results For semi quantitative analysis of protein expressions array staining can be detected with en zyme conjugated secondary antibodies and visualized via color reaction For example one may use alkaline phosphatase conjugated secondary antibodies in the following manner 1 After array staining place the coverslip in a washing container Remember the orien tation of the coverslip in the container 2 Wash with PBS three times 3 Apply alkaline phosphotase conjugated goat anti rabbit and goat anti mouse secon dary antibodies to the cells 4 Incubate for 1 hour 5 Wash with PBS three times 6 Add phosphotase substrate 3 bromo 4 chloro 5 indolyl phosphate nitro blue tetra zolium BCIP NBT 7 Incubate until a brown color develops When proper intensity is reached stop the reaction by washing the substrate with PBS The image of the StainingArray can be scanned on a stan
19. tions should be carefully examined before experiments Testing Staining AntibodyArray can be used for this purpose 2 One cell fixation condition may be optimal only for the staining of a subset of the proteins Therefore several conditions may be needed to obtain proper staining for all the antigens 3 The following protocols are for reference only Researchers should carefully study their systems and select proper experimental conditions B Protocol for AntibodyArray staining 1 Fix and permeabilize cells to exposure antigens for detail see Protocols D to D 2 Block staining AntibodyArray and fixed cells in PBS solution containing 5 BSA for 0 5 1 hour 3 Place the staining AntibodyArray on top of the cells and assemble the components as shown in Fig 4 Screw lt 1 Moving plate Pad Pad Filter paper lt lt Staining AntibodyArray AAL Fixed cells Filter paper lt Apparatus frame Fig 4 Assembly of various staining components 4 Apply pressure and secure the assembly 5 Stain for 1 to 2 hour 6 Remove the array from the cell support and put the cells in a container 7 Rinse the cells with PBS www hypromatrix com 6 Hypromatrix Inc AntibodyArray Instruction Manual 8 Apply fluorescence labeled secondary antibodies a mixture of anti rabbit and anti mouse antibodies and incubate for an hour 9 Wash with PBS 10 Visualize the staining under fluorescent micro
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