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Dear Tech Support Team,
Please see below the details of our customer's complaint.
It is important to emphasize that this ab was supplied as the replacement for ab65815 that also did not perform well (the correspondence is attached as well).
Antibody code: ab103679
Batch number: GR29484-5
Antibody storage conditions Stored at 4C for short period (aliquots in -20C)
Description of the problem No specific staining
Sample Parrafin sections of human and mouse testis, and rat ovaries
Fixation of sample Paraformaldehyde 4% for 24 hours
Antigen retrieval Heat induced, tried both citrate pH 6.0 and TE pH 9.0
Blocking conditions Blocked in buffer containing 0.3% Triton, 0.05% Tween-20 and 4% Normal Donkey Serum. Also tried same buffer without Triton. Blocked for 1 hour.
Primary Antibody Diluted antibody 1:50 or 1:100 in blocking buffer, incubated overnight at 4C. Washed with PBS.
Secondary Antibody Biotin conjugated antibody from Jackson immunoresearch. Diluted 1:200 in blocking buffer.
Detection method Cy2 or Cy3 conjugated avidin
Positive and negative controls used Other antibodies stained the same sections with no problem. No negative control as the issue is lack of signal
Optimization attempts (problem solving):
How many times have you tried the IHC? 6
Have you run a "No Primary" control? Yes – no issues.
Do you obtain the same results every time? Yes
What steps have you altered? I have altered the antigen retrieval buffers, and tried removing triton from the blocking buffer. Also tried different dilutions of the antibody
SF1 is expressed in the steroidogenic cells of the testis (Sertoli) and ovaries (Granulosa). These cells are present in the tissue, and stain positive for other markers (ie Sox9 with abcam ab76997), but there is no staining whatsoever.
Document Attachment: images of the results may be very helpful. If you wish to add it to the complaint form, please attach them to the reply e-mail.. ATTACHED
Please read below customer's additional comments:
Does abcam have any other primary antibody against SF1 good for IHC in human?
This is the third out of 4 antibodies I have tried for this project that is failing. I am beginning to worry that maybe abcams QC isn't what it used to be.
The slides and reagents I use are obviously expensive, and it is frustrating to have to waste so many in an attempt to get any kind of staining. I always add a positive control to my experiments, so I know that both the slides and the assay work.
Thank you for your assistance and reply.
Asked on Aug 15 2012
Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody and that you have had difficulties with previous products aswell.
The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.
I would like to reassure you that ab103679 is tested and covered by our 6 month guarantee for use in IHC-P and human samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.
Reviewing this case, I would like to offer some suggestions to help optimise the results. I would also appreciate if you can confirm some further details:
1. I fully understand your concerns and it is highly unusual for 4 antibodies not to work for a customer. In this case, I can suggest to consider reviewing the sample preparation in particular. For example, try 1 fixing in 10% PFA for at least 24 hours.
2. Antigen retreival can often require individual optimization for different antibodies with different epitopes. For example, I can recommend to try different time points for antigen retrieval. Try 2, 3, 5, 10 and 20 minutes.
3. As the target protein is nuclear, I can recommend to include a separate permeabilization step before staining.
4. I would suggest to try a lower concentration of antibody, 1:200 or even 1:500, to try and reduce the background.
5. I can recommend to include 0.2% Tween in wash buffer to help keep the cells permeabilized and to wash away any non specific binding. Secondary antibody working well with other primary antibodies?
6. 0.3% Triton is quite high, and may affect the staining. Try 0.2%.
I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.
Answered on Aug 15 2012