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I ordered Anti-BLBP antibody - Neuronal Marker (ab32423) a couple of weeks ago and the IHC did not work.
I used the following protocol:
1.Mouse embryos were fixed with 4%paraformaldehyde, 4 C, 1h
2. embedding in agarose and sectioning
3. wash with 1X PBS, 10mins x2
4. 0.1% triton/PBS, RT 20mins
5. 5% heat-inactivated Goat serum/0.1%/PBS 1hr
6. 1st antibody RT 2hr (1:200, in 5% heat-inactivated Goat serum/0.1%/PBS)
7. wash with 0.1% triton/PBS, 10 mins x2
8. 2nd antibody 1h(1:500, in 5% heat-inactivated Goat serum/0.1%/PBS)
9. wash with 1X PBS 10 mins x2
Could you please help me to figure out what is the problem?Thanks,
Asked on Nov 19 2012
Thank you for contacting us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.
I would like to investigate this particular case further for you, and also obtain more information for our quality records. In order to proceed with this, there are a few things I would like to clarify to better understand and solve the problems experienced with this antibody.
Could you please describe the problem experienced with the antibody? Didn’t you observe any signal at all, unspecific signal, high background? I would appreciate if you could please provide an image of the tissue sections in order to better understand the problem described.
Also, the order number and / or lot number is also important to dismiss shipping failure and monitor our product’s quality. Could you please provide this information?
Regarding the protocol followed, was the antigen retrieved after fixation? If so, which method was used? Was the secondary conjugated to an enzyme or was it a fluorometric method?
Unfortunately, this antibody has only been tested with frozen and paraffin embedded sections, we do not have any evidence to support its performance in agarose embedded sections. In order to assess the antibody’s activity, I would suggest trying a protocol which has been proven to give positive results in our laboratory. If you had frozen or paraffined sections available, I would suggest giving it a try.
Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), and if it appears that the antibody is at fault, a replacement/credit note/refund will be offered.
Thank you for your comprehension. I look forward to receiving your reply.
Answered on Nov 19 2012