Question (15963) | Anti-IL-8 antibody (ab7747)

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Question

Sorry about that - here it is. Primary antibody: ab7747 Secondary antibody: Biotinylated goat anti-rabbit IgG [a competitor] Chromogen: AEC Procedure: 1. Place slides on hot plate, 65 C for 30 minutes, deparaffinize in xylenes 4 min x 2, hydrate through alcohol –100% 2 min x 2, and 95% alcohol 2min x 2, 80% alcohol 2min x 1, then wash with PBS 3 min x2. 2. Perform antigen retrieval: Dako target retrieval buffer. Place slides in coplin jar filled with 1X antigen retrieval buff (DAKO). Pre-heat buffer for 20 minutes in steamer. Add tissue for 20 min, then leave in steamer to cool down for 30 min. 3. Draw a hydrophobic circle around the tissue with a pap pen. Add a drop of PBS and leave in humidity chamber. 4. Block endogenouse peroxides with 3% H2O2 in PBS for 12 min. 5. Wash with PBS 3min x 3. 6. Incubate with protein clocking solution (5% normal horse serum + 1% normal goat serum in PBS) 20 min. 7. Remove excess protein block soln, and add primary antibody in protein block solution (see antibody dilution schedule) and incubate overnight at 4 C. 8. Day 2, wash with PBS 3min x 3. 9. Add protein block solution and incubate for 10 min. 10. Remove excess protein block soln, add secondary antibody: biotinylated goat anti-rabbit IgG (Biocare Medical) 30 min, 11. Wash with PBS 3min x3 12. Strepavidin conjugated with horseradish peroxidase (Dako) in PBS (1:300) for 30 min. 13. Wash with PBS 3min x 3, followed by a brief rinse in dH2O with brij (1 drop of brij in 50 ml of PBS) 14. Add chromogen (AEC) and incubate for 2-5 min at RT. Check the reaction microscopically to determine the end point. 15. Wash with dH2O and dry on heat 65 C for 10-20 min, then mount with universal or Permont (Fisher) mounting solution. Leave slides overnight for a complete dry.

Answer

Thank you for your protocol, I have looked at it in details and I would like to suggest trying the following changes to see if you start having a cytoplasmic staining rather than a nuclear staining. -check you are not overfixing (or underfixing) the tissue, different fixation times could be tested to see if this may be a problem -try a different antigen retrieval method, such as enzymatic. This way if the citrate buffer antigen retrieval method is damaging the epitope (and other proteins) you may see a difference with a different antigen retrieval step -check with a no primary control that the staining is not due to the secondary or other further steps in the staining protocol -try blocking peroxidases with only 0.3% 10-20min, too much peroxidase can damage the tissue. -remove the horse serum and use only 10% normal goat serum to block the tissue (just in case the serum is giving the problem) I think in general your protocol is very good and hope that the above suggestions will help. If you want to be certain of the staining I would recommend to run another IL8 antibody along the sister sections to see if it gives you the same staining pattern. As ab7747 has not been tested in IHC it is hard to be sure the problem is not that the antibody does not work in IHC.

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