Multiple Antigen Labeling - Vector Laboratories

More documents

Recommendations

Info

14Immunofluorescence Staining MethodsImmunofluorescence staining methods can alsobe used successfully for labeling multiple antigensin the same preparation. These methodsare especially useful for co-localization of antigensin the same compartment of a cell, and inthis regard offer a distinct advantage overenzyme-based detection systems.Traditionally, double fluorescent labeling hasbeen achieved by using fluorescently conjugatedsecondaries against primary antibodies fromdifferent species. Although this technique ingeneral tends to be less sensitive than enzymaticstaining, the sensitivity of the fluorescent staincan be increased by using a (strept)avidin-biotinsystem for amplification of the label. Many of theprinciples regarding multiple antigen labelingwill apply to both immunofluorescent andenzymatic methods (see section on “ImmunoenzymaticStaining Methods”). For best results,sequential staining of each primary antibody isrecommended. As in enzymatic applications, theorder of labeling, appropriate controls, andadditional blocking steps may be important toobtain optimal results. In contrast to enzymaticstaining, special consideration must be given to thespecies of the primary antibodies to be used. Forfluorescent applications, the two primary antibodiesusually should be from different species,otherwise, artifactual co-staining of antigens mayresult. However, the use of two mouse primaryantibodies is the exception. See section on“Multiple Immunofluorescent Labeling Using Twoor More Mouse Monoclonal Primary Antibodies”.In addition, if the (strept)avidin biotin system isused for the visualization of both antigens, a(strept)avidin-biotin block MUST be used beforethe second primary antibody step.Following labeling it is important to preservethe intensity of the fluorescent signal, as somefluorescent products are prone to rapid fadingwhen viewed under the microscope. In mostinstances this is easily accomplished by coverslippingthe preparation with an anti-fade, antiphotobleachingagent such as VECTASHIELD ®mounting media. The use of VECTASHIELD ® alsoallows the preparation to be stored for extendedperiods without significant loss of intensity.Autofluorescence of the tissue may obscurestaining depending on the type of tissue, thefixation method, and the microscope filter usedfor visualization. This should be evaluatedbefore staining, and autofluoresence quenched,if necessary, using appropriate methods.The following protocols are for frozen tissuesections. These protocols can be adapted forother tissue preparations.Colon (frozen) – Double label• Multi-Cytokeratin (m), M.O.M. Fluorescein Kit (green).• Desmin (m), M.O.M. Basic Kit, Texas Red ® Avidin DCS (red).Tonsil (frozen) – Double label• Ki67 (m), M.O.M. Fluorescein Kit (green).• Pan-Cytokeratin (m), M.O.M. Basic Kit, Texas Red ® AvidinDCS (red).Small Bowel (frozen) – Double label• PGP9.5 (m), M.O.M. Basic Kit, VECTASTAIN ® ABC-APStandard Kit, Vector ® Red AP substrate (red).• Desmin (m), M.O.M. Fluorescein Kit (green).www.vectorlabs.com
15Protocol: Double Immunofluorescent Labeling Using Two PrimaryAntibodies From Different SpeciesStaining for First Antigen1. Preparation of tissue. Fix sections with theappropriate fixative for the antigen under study(Please see Note 1).2. Air dry sections.3. Wash sections 2 x 2 minutes in buffer (PBS).4. Avidin/biotin blocking step. PerformAvidin/Biotin blocking if required (Avidin/BiotinBlocking Kit, Cat. No. SP-2001). Incubate sectionswith Avidin Solution for 15 minutes. Rinsebriefly with buffer, then incubate in the BiotinSolution for 15 minutes. Wash sections 2 x 2minutes in buffer. This blocking step may beeliminated if suitable controls have determinedthis step to be unnecessary.5. Protein blocking step. Incubate sections for20 minutes with buffer containing 5% normalblocking serum (NS) which was prepared from thespecies in which the secondary antibody is made.6. Blot excess serum from sections.7. Primary antibody. Incubate sections with thefirst primary antibody diluted in appropriate antibodydiluent (buffer containing 5% NS) using anappropriate concentration and length of incubation.8. Wash for 5 minutes in buffer.9. Secondary antibody. Incubate sections for30 minutes with biotinylated secondary antibody(5-10 µg/ml diluted in buffer containing 5% NS).10. Wash slides for 5 minutes in buffer.11. Avidin conjugate. Incubate sections withFluorescein Avidin DCS (15-20 µg/ml diluted inbuffer) for 5-10 minutes.12. Wash slides for 5 minutes in buffer.Staining for Second Antigen13. Avidin/biotin blocking step. Apply anAvidin/Biotin block according to Step 4. (Thisstep must be done to prevent the interaction ofthe second set of labeling reagents with thefirst set of labeling regents).14. Protein blocking step. Incubate sections for20 minutes with 5% NS.15. Blot excess serum from sections.16. Primary antibody. Incubate sections withsecond primary antibody diluted in appropriateantibody diluent (buffer containing 5% NS)using an appropriate concentration and lengthof incubation.17. Wash slides for 5 minutes in buffer.18. Secondary antibody. Incubate sections for30 minutes with biotinylated secondary antibody(5-10 µg/ml diluted in buffer containing 5% NS).19. Wash slides for 5 minutes in buffer.20. Avidin conjugate. Incubate sections withTexas Red ® Avidin DCS (15-20 µg/ml diluted inbuffer) for 5-10 minutes.21. Wash slides for 5 minutes in buffer.22. Mount with the appropriate VECTASHIELD ®mounting media.23. Observe under a fluorescence microscope.NOTES:1. Aldehyde-fixed tissues (e.g. formalin) tend tobe autofluorescent and may make interpretationof specific fluorescein signal difficult.12 3 4Add first primary antibody.Add biotinylated secondaryantibody.Add Fluorescein Avidin DCS.Add avidin/biotin block.5 6 7 8Add second primary antibody.Add biotinylated secondaryantibody.www.vectorlabs.comAdd Texas Red ® Avidin DCS.Mount and observe.
Page 1 and 2: DISCOVERYthrough color.A Guide to M
Page 3 and 4: 1Multiple Antigen Labeling in the S
Page 5 and 6: 3Peroxidase SubstratesIn general, p
Page 7 and 8: 5Order of Substrates in Labeling Pr
Page 9 and 10: 7Fig. 5Fig. 6Fig.7Add Second Primar
Page 11 and 12: 9General NotesControl SectionsContr
Page 13 and 14: 11Tonsil - Double label• CD3 (m),
Page 18 and 19: 16Multiple Immunofluorescent Labeli
Page 20 and 21: 18Skeletal Muscle (frozen, unfixed)
Page 22 and 23: 20Appendix 2: Quenching Endogenous

Multiple Antigen Labeling - Vector Laboratories

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?