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Dear Tech Support Team,
Please read below the details of the customer's complaint and advise.
Thanks in advance for your reply.
Order Details: Donkey anti-Mouse IgG H&L (DyLight® 488) secondary antibody
Antibody code: ab98794
Antibody storage conditions +4oC
Description of the problem non-specific staining
Sample: Brain of rats
Fixation of sample Methanol
Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
Blocking conditions PBS+8% normal donkey serum at 23oC for 60 min
Primary Antibody Dilution between 1:100-1:500 on PBS+ 1.3% NDS over night at 4oC
Secondary Antibody Donkey anti-Mouse IgG H&L (DyLight® 488) diluted 1:500-1:1000 in PBS+NDS incubation at RT for 120 min
Positive and negative controls used We are working on frozen sections and we have tried double immunostaining with another anti rabbit secondary and we got excellent results with the anti rabbit and no signal with the Donkey anti-Mouse secondary. We also run this secondary antibody in another set of sections with a primary antibody that we know for certain that works. In one set the GR primary was run with the donkey anti mouse and in the second with goat anti mouse-Dylight -594 (from KPL). We got no results with the donkey-anti mouse and good results with the goat anti-mouse.
Optimization attempts (problem solving):
How many times have you tried the IHC? Several times with different primary antibody dilutions
Have you run a "No Primary" control?
Do you obtain the same results every time? Yes there is no signal
Asked on Jul 17 2012
Thank you for your enquiry regarding ab98794 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.
It is clear that the protocol worked fine and an alternative secondary antibody provided the expected results. The image you have kindly sent to me showed the significant difference in the staining pattern/quality.
Could you please confirm the batch and order number. Would your troubled customer prefer to get a new vial (free of charge replacement) or a credit note?
I look forward to hearing from you soon.
Answered on Jul 17 2012