Anti-CD3 epsilon antibody, prediluted (ab27434)

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 175529

DESCRIPTION OF THE PROBLEM This antibody binds non-specifically, we noted nuclear staining.

SAMPLE Mouse' brain and spleen tissue.

PRIMARY ANTIBODY Abcam rabbit-CD3 prediluted antibody abab27434. Already comes prediluted. Tissues were incubated for 10min or 30 min. We also tried 1 hr and overnight.

DETECTION METHOD Used DAB and also flourescence (Alexa).

POSITIVE AND NEGATIVE CONTROLS USED We used spleens as positive controls and found extensive non-specific binding. We also included some negative controls (no primary).

ANTIBODY STORAGE CONDITIONS 4 degrees as recommended by the manufacturer.

FIXATION OF SAMPLE Tissues were fixed in 4 % PFA. Incubated in methanol/H202 for 20 min prior to initiating washes in dH20 followed by PBS 1%. Tissues were paraffin embedded.

ANTIGEN RETRIEVAL Tissues were heated to 99 degrees in either citrate buffer or EDTA. Tissues were allowed to cool down to room temperature before proceeding with washes.

PERMEABILIZATION STEP No permeabilization step was used to protect surface antigens.

BLOCKING CONDITIONS Used normal goat serum (5%) with PBS 1% to block and with primary and secondary antibody. We also tried BSA (3%) with NGS/ PBS. We blocked for 30 min or 1 hr before adding primary.

SECONDARY ANTIBODY The secondary we used was labelled polymer HRP anti-rabbit. One dropp (100 ul) were added to each tissue and incubated for 1 hr. Tissues were washed with PBS 1% 3X for 5 minutes

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

WHAT STEPS HAVE YOU ALTERED? We altered the time of incubation for the primary. We also tried EDTA instead of citrate buffer for antigen retrieval. We also tried

ADDITIONAL NOTES We specifcally purchased this antibody because it should have worked on embedded tissue. We also find that it does not work on frozen sections as we tried that as well. At this point we've spent more time trying to resolve this issue and have not made any progress. We are extremely unhappy with your product and the handling of this matter.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. From your previous correspondence I can see that you have contacted us on several occasions in recent months. I am also sorry for the delay to your enquiry and a resolution to this matter.

I have read your technical questionnaire and I have a few comments. You are largely applying the antibody in a manner that I would recommend. However, please can you detail the duration and pH of the antigen retrieval approach that you are using in addition to the effect of changing the antigen retrieval solution to EDTA; whether this improved or changed the nuclear or cytoplasmic distribution of the antibody staining.

I thank you for your patience and look forward to your response.