2011-2012 Catalogue - PeproTech, Inc.

PEPROTECH ®www.peprotech.comˇProblem Possible Source Suggestionˇ Antigen • This antigen may not be present in the particular tissue or at aˇsignificant level for detection.ˇ• The epitopes of the antigen may have been destroyed duringˇtissue processing or antigen retrieval. Also, if using a monoclonalˇantibody and that particular epitope is masked or destroyed, thenˇthe antibody will not stain. Consider re-staining or reprocessing aˇnew tissue sample.ˇ• Use an alternative assay to detect for the presence of the antigenˇ(e.g. in situ hybridization).ˇ Chromogen • Adjust incubation time. Check for chromogen precipitate and doˇnot use past expiration date.ˇ• Use appropriate mounting media for each chromogen (e.g. DABˇcan be coverslipped with a permanent mounting media, while aˇnon-alcohol soluble AEC will require an aqueous mounting media).Over-staining Tissue Processing • Ensure tissue is prepared properly prior to immunostainingˇprocedure. Use proper fixative, fixation time, embedding reagentsˇand embedding techniques. Use proper sectioning techniquesˇand equipment. The recommended tissue thickness for IHC isˇ4µm sections. For formalin-fixed, paraffin-embedded tissue,ˇensure slides are completely deparaffinized and rehydrated.ˇReplace xylenes and alcohols as needed.ˇ Antigen Retrieval • The tissue may be over-retrieved. Adjust the incubation time.ˇAdjust the temperature. If necessary, use alternate antigenˇretrieval methods. Stain a tissue section without antigen retrieval.ˇ Primary Antibody • Ensure antibodies are prepared as per data sheet:ˇ• Centrifuge vial prior to opening.ˇ• Once the antibody is reconstituted, centrifuge vial to remove anyˇpossible aggregates.ˇ• Aliquot and freeze antibody at -20˚C. Avoid repeated freeze/thawˇcycles.ˇ• Use antibody before expiration date.ˇ• Decrease antibody concentration. Adjust antibody incubation time.ˇ• Incubate tissue specimen with a protein block just prior to theˇprimary antibody incubation (see protein block below).ˇ Secondary Antibody • Adjust concentration of secondary antibody. Use a secondaryˇantibody that is less likely to exhibit non-specific binding (e.g.ˇcross-adsorbed antibody or F(ab’) 2fragment).INFORMATION & TECHNICAL SUPPORTˇ Detection System • Use a detection system that will exhibit less non-specific binding.ˇ• Adjust incubation time(s).ˇ Non-specific Binding • Block endogenous molecules and highly charged molecules.ˇ Endogenous • Depending on the tissue type and staining procedure, endogenousˇ Molecules molecules may be present and interfere with staining. Determineˇthe necessary blocking steps for each tissue, such as blocking forˇendogenous molecules such as endogenous biotin, endogenousˇperoxidase, endogenous phosphatase, or Fc Receptors.ˇ• Choose an alternative staining method when an endogenousˇmolecule may be difficult to completely block (such as a non-ˇbiotin detection method on liver, which is rich in biotin).Bestellungen: Deutschland: Tel: 0800 436 99 10 • email: info@peprotech.dePour commander: France: Tel: +33 (0)1 46 24 58 20 • email: info@peprotechfr.comTo Order: NORDIC: Tel: +46 (0)8 640 41 07 • email: info@peprotech.se175