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1 question for ab81212
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Question 1
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Thursday 26-January-2012 |
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Dear Technical
Order Details:
Antibody code: ab81212
Batch number: GR62732-1
General Information:
Antibody storage conditions (temperature/reconstitution etc)
Stored at -20C.
Description of the problem (high background, low signal, non-specific staining etc.)
No staining and background.
Sample (Species/Tissue/Cell Type/Cell Line etc.)
1. Human Breast cancer.
2. Mouse liver implanted with human cells.
Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
Paraformaldehyde for about 2 days at room temp.
Prepared paraffin embedded slides from the sample.
Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
Heat mediated Citrate buffer.
Permeabilization step
Blocking conditions (Buffer/time period, Blocking agent etc.)
Primary Antibody (Diluent/Dilution/Incubation time, Wash step)
Diluted at 1:1000 and 1:300 in Invitrogen green antibody diluent, Cat # 00-3118.
Incubated for 1 hr at room temp.
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Super Picture Ploy HRP conjugate from Invitrogen, Cat # 87-8963.
Detection method
DAB staining
Positive and negative controls used (please specify)
Positive control – Human breast cancer
Optimization attempts (problem solving):
How many times have you tried the IHC?
1
Have you run a "No Primary" control?
No
Do you obtain the same results every time?
Yes
What steps have you altered?
Additional Notes
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.. |
ANSWER: |
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Thank you for your enquiry regarding ab81212 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody.
The protocol looks fine to me, however it would be much appreciated if I could get some more information which would help me identify the source of the problem.
1) Antigen retrieval: I understand that one experiment has been carried out only and the result is negative. Could you please let me know how long and at what pH the demasking was performed? Has any optimization been tried?
2) Incubation: Has the customer tried longer incubation such as 2 hrs or overnight to see if the signal is getting stronger at all?
3) Permeabilization: mtND6 (NADH Dehydrogenase subunit 6) is localized in the Mitochondrion membrane so permeabilization of the membrane may be important. I am not sure what the green antibody diluent contains?
4) Detection system: Is the secondary antibody compatible with the primary antibody (rabbit IgG)? Could you please confirm? Does the detection system work fine? Have you used it successfully with another primary antibody? Is the HRP still active?
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible. |
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