Anti-PAX5 antibody (ab2935)

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 211218

DESCRIPTION OF THE PROBLEM Non-specific staining of tissue

SAMPLE Human tissue, tonsil. 4 and 10 µm sections mounted on positively charged slides.

PRIMARY ANTIBODY Ab2935 - Goat anti-human - diluted in the following dilutions: 1/20, 1/25, 1/50, 1/100, 1/250, 1/500, 1/1000. Antibody incubation was tried at 30 minutes (standard) and 1 hour at room temperature and overnight at 4°. Washing was done in buffer bath for 3 X 5 minutes with both homemade Tris buffer (pH 7) and Dako Tris buffer for IHC. Staining was tried both manually and on Dako autostainer.

DETECTION METHOD Detection was done using Dako's envision plus rabbit kit, and DAB+ chromagen, as per Dako dilution instructions.

POSITIVE AND NEGATIVE CONTROLS USED Positive control tissue - human tonsil. Known to express Pax-5 with other Pax-5 Ab IHC staining (done at same time)

ANTIBODY STORAGE CONDITIONS Stored at -20 until thawed. After thawing, Ab was kept at 4 degrees and not diluted until immediately prior to use.

FIXATION OF SAMPLE Formalin fixed parrafin embedded tissue (by standard methods), tissue sectioned onto slides, immediately baked at 60 degrees overnight and cooled to room temperature before IHC.

ANTIGEN RETRIEVAL Tried the following methods with no success: Microwave heating of slides for 20 minutes (followed by 20 minutes of cooling to RT in heated buffer) in the following: Citrate buffer, pH 6 - no staining at all CItrate buffer, pH 10 - non-specific staining at lower dilutions EDTA buffer, pH 9 - non-specific staining at lower dilutions Enzymatic method - protease, following regular pH 9 HIER for 4 minutes prior to blocking and IHC- no change in stain quality

BLOCKING CONDITIONS Blocking - prior to HIER, following deparrafinization, 3% hydrogen peroxide in methanol to block endogenous peroxides prior to final hydration. Following HIER, sample was blocked with 3% Hydrogen peroxide block solution, part of the Dako envision plus IHC staining kit.

SECONDARY ANTIBODY Dako rabbit anti-goat, diluted to 1/500, 1/1000, 1/2000 in Dako antibody diluting buffer for 30 minutes and 1 hour. Washing was done in buffer bath for 3 X 5 minutes with both Tris buffer (pH 7) and Dako Tris buffer for IHC. Staining was done both manually and on autostainer. The Dako Envision plus klit for detection of rabbit antibodies was used as the final antibody for detection, at the prediluted concentrations in the kit.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10+ HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? pH of HIER buffer, antibody concentrations, antibody incubation times, incubation temperatures, rinsing buffers, different tissue blocks, both manual and automated.

ADDITIONAL NOTES We purchased this Ab specifically to do a blocking study and were told to try it. It does not give us any results that can be useful to us. The staining we get is non-specific and muddy when compared to another Pax-5 antobody successfully used previous in the lab with the same methods and tissues. This Ab was specifically purchased for blocking studies using Ab22941 peptide

ANSWER:

Thank you for your patience.

I am sorry to hear that you are experiencing difficulties with this product ab2935 in immunohistochemistry. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support but please bear in mind that this product has not been tested in IHC application and therefore, might not be suitable for use.

I have looked at the protocols you used and I have questions regarding the Dako’s envision plus rabbit kit. Are you using this as amplification to your secondary antibody? Do you have a rabbit anti-goat HRP secondary antibody that you can use to detect the primary antibody? I am concern that the no staining problem may lie with the secondary/tertiary antibody.

If you have used the same protocols and materials with the other Pax-5 antibody and did not get results, then I am afraid that this product is indeed not suitable for use in IHC application.

In any case, can I encourage you to submit an Abreview via the online product datasheet? We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to other researchers. We reward each Abreview with Abpoints which can be used for discounts off future purchases and gifts. If you decide to submit an Abreview, please include a note that you have already spoken to me, so that I will be able to quickly publish your Abreview, as I have already tried troubleshooting with you.

To find out more about our Abreview system, please see the following URL: http://www.abcam.com/abreviews

Should you require any further information or assistance, please do not hesitate to contact me.