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My customer did not consent to giving you her data, I suppose that she afraid to talk in English, she may not feel confident but look at her answers/more details below:
1.deparaffining scrap-xylene, alcohol, H2O
2 - Reveal antigen; a) hot - citrate buffer pH 6-20 minutes
b) hot-EDTA, pH -9
c) the enzyme - trypsin-Sigma
3 - Cooling the room at temperatures - 20 minutes
4-Methanol + H2O2 - 10 minutes
5 - Tris buffer pH 7, 6 - 10 min
6 - Blocking serum (NCL-H-SERUM)-Leica-20 min.
7 - the primary antibody ab 20 384 - 5ug / 1 ml, room temperature overnight and humid conditions
8-Tris buffer pH 7, 6 - 10 min
9 - Secondary Antibody Anti-Mouse IgG-Sigma - B 7151 (1:200) - 45 min
10-Tris buffer - 10 min
11 - Streptavidin-HRP Conjugate Millipore (1:500) 60 min
12-Tris buffer, 10 min
13 - DAB - 1 to 3 min
14 - Hematoxylin - coloring
15 - Dehydration and seal in histofluid.
The problem is the lack of an immune response each time revealing.
The positive control is the human tissue in Alzheimer's patients.
Reagents that were used for staining routinely used for staining with other antibodies without problems
Asked on Jul 19 2012
Thank you for taking the time to provide the further information.
The details you have kindly provided will provide us with vital information for our monitoring of product quality
I appreciate the time the customer has spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial. I apologize for the inconvenience.
I have also spoken to our distributors team and I am happy to let you know we are prepared to make an exception and continue to provide our Abpromise for Symbios customers. Therefore, I am pleased to offer you a free of charge replacement or credit note in compensation for this case.
In addition, I can suggest the customer may like to consider the following suggestions as a check for future experiments:
1. I can suggest to try adding 0.2% Tween to the antibody dilution buffer and wash buffer. This will help to solubilise the antibody and keep the cells permeabilised.
2. I can recommend to try wash steps: 3 X 5 minutes in PBS containing 0.2% Tween.
3. We recommend to incubate at 4oC overnight with antibodies. This can often provide more efficient and specific staining.
4. Is the current vial of secondary antibody working well with other primary antibodies?
I hope this will be helpful. I look forward to hearing from you with details of how you would like to proceed.
Answered on Jul 19 2012