Anti-Survivin antibody [8E2] (ab3258)

DESCRIPTION OF THE PROBLEM Non-specific staining

SAMPLE Human/stomach

PRIMARY ANTIBODY Survivin ab3258/mouse/pbs/bsa /1/75 /4n/o/tbst

SECONDARY ANTIBODY Chemicon select kit Secondary Antibody

DETECTION METHOD DAb

POSITIVE AND NEGATIVE CONTROLS USED Tumour tissue +

ANTIBODY STORAGE CONDITIONS 4dgrees

FIXATION OF SAMPLE Formalin

ANTIGEN RETRIEVAL Heat mediated

BLOCKING CONDITIONS Chemicon select kit block

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Ab dilution

1. At what dilution(s) of the primary antibody have you tested? You have mentioned that you tried several dilutions but no more details about it?

0,1/50,1/75,1/100,1/250,1/500

2. Can you specify the secondary antibody, please? Is it Anti-mouse and in which species was it raised? Is the secondary antibody compatible with the primary antibody? Have you used it with another antibody? Does the detection system work properly? Is the HRP still active?

Biotinylated goat anti-mouse IgG and goat anti-rabbitIgG. HRP still active in other expt.DAB still reactive as demonstrated using other abs.

3. This antibody has been tested for IHC using HeLa cells. Have you used positive control? Yes a gastric tumour.

4. What sort of blocking agent did you use? Milk, serum, BSA (concentration, time, temperature)? Normal goat serum in PBS

5. How long have you done the heat mediated antigen retrieval? In which buffer? Could you please a bit more specific? There is a possibility that the blocking step has not been sufficient.

10mM Na citrate buffer pH 6.0,slides brought to boiling,then sub -boiling for 10mins,cool at R.T for 30mins

Hope this answers your Q's

ANSWER:

Thank you for getting back to us and providing further details of your IHC staining.

We have looked through your e-mails and it seems that the protocol is absolutely fine. We have searched our database and found that this is a popular selling product and your feedback is the first we have received about it not working. Therefore, at this stage, we would suggest that there is probably a problem with the vial you received.

We can offer you a new vial free of charge. If you wish to get it, could you please confirm your Abcam Order Number and your Purchase Order Number.

We look forward to hearing from you soon.

My customer would need a Survivin antibody to use it in FACS. SHe's not sure whether to use ab4470 or ab3258, what can I suggest her?

ANSWER:

Neither of the antibodies have been tested in FACS therefore I cannot recommend either without further information.