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Inquiry: 1)Antibody storage conditions (temperature/reconstitution etc) -20 °C 2)Description of the problem (high background, low signal, non-specific satining etc.): Non-specific staining and no specific signal 3)Sample (Species/Tissue/Cell Type/Cell Line etc.): Rat Brain (hippocampus) 4)Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.): Paraformladheyde perfusion followed by immersion and sucrose embedding. Frozen 40 micron sections 5)Antigen retrieval (Enzymatic method, Heat mediated technique etc.): No 6)Permeabilization step: Standard with 0.1% Triton in PBS 7)Blocking conditions (Buffer/time period, Blocking agent etc.): 2.5% normal goat serum in PBS-Triton 8)Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step): ab 18723, diluted 1:1000 in blocking buffer, overnight incubation in the cold room. 9)Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step): Anti-rabbit HRP conjugared Santa Cruz or anti-rabbit conjugated with Alexa fluor 568, Invitrogen for immunofluorescence, 2 hour incubation ar room temperature. 10)Detection method: DAB for HRP conjugated secondary antibody 11)Positive and negative controls used (please specify): Positive control, differentiating neuroblasts in juvenile hippocampus. Negative control differentiated neuron in adult hippocampus. 12) How many times have you tried the IHC?: Many times with different lots of the same antibody in the past with good results and 4 times with lot GR 55971-1 with bad results. 13)Have you run a "No Primary" control?: No 14)Do you obtain the same results every time?: Yes 15)What steps have you altered? None Additional Notes We have used for more than one year different lots of the same antibody (no bottle conserved) with satisfactory rwsults (see figures marked as old ab). Storage conditions in house, protocol and material used have been always the same.
Asked on Apr 19 2012
Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.
The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.
Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.
I apologize for the inconvenience and am pleased to offer you a free of charge replacement with a different lot of ab18723. Or if you would prefer a credit note. If you would like for me to organise this could you please confirm the order number (or purchase order number) of ab18723.
Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.
Answered on Apr 19 2012