Anti-MSH2 antibody [3A2B8C] (ab52266)

LOT NUMBER GR11201 ORDER NUMBER 817821

DESCRIPTION OF THE PROBLEM No staining

SAMPLE Human paraffin embedded sections

PRIMARY ANTIBODY 1:1000 Golden Bridge International Inc. Antibody Diluent 1h 30min incubation

DETECTION METHOD LSAB

POSITIVE AND NEGATIVE CONTROLS USED -

ANTIBODY STORAGE CONDITIONS -20C

FIXATION OF SAMPLE fixative agent : 10% neutral buffered formalin fixation time : 1 day fixation temperature : RT

ANTIGEN RETRIEVAL EDTA buffer pH8.6

PERMEABILIZATION STEP -

BLOCKING CONDITIONS -

SECONDARY ANTIBODY Goat anti-Rabbit (Universal kit) 30min incubation

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? antigeb retrieval step -->citrate buffer, pH7.0

ADDITIONAL NOTES -

ANSWER:

Thank you for your enquiry regarding ab52266 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody. Additionally, thank you for supplying an image, as this has been very beneficial in understanding your concerns.

I would like to ask few questions which would help me identify the source of the problem;

- What is the type of human tissue used? Does the tissue express this target naturally? - In the provided files there are two set of images. What these images means? Which image related to ab52266?

The protocol looks fine to me however I would like to make few comments/suggestions that might help to improve our results and that you could consider trying:

- The protein is nuclear so we would recommend permeabilizing the tissue section either by incubating the section with 0.25% Triton X-100 or by mixing 0.25% Triton with antibody incubation buffer. - If the first line of images is with ab5266 then it looks like the antibody is binding non specifically. I would recommend trying more diluted primary antibody e.g. 1/1500 or 1/2000. It will help in optimization of protocol. - I would recommend blocking the tissue sections with goat serum for 1 - 2 hours at room temperature. - Could you specify which antigen retrieval method is being used; microwave or pressure cooker? We recommend optimizing the method by incubating the sections at different intervals of time. e.g. for microwave we recommend incubating sections for 5, 10, 15, 20, 25 and 30 minutes and for pressure cooker 1, 2, 3, 4 and 5 minutes, to evaluate optimum antigen retrieval time for the particular antibody being used.

I hope these suggestions will help to improve the results. Please do not hesitate to contact if you have any further questions.