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LOT NUMBER GR18150
DESCRIPTION OF THE PROBLEM Non-specific staining
SAMPLE Species Human Type of sample: paraffin embedded sections
PRIMARY ANTIBODY Concentration or dilution:dilute 200X and 50X Diluent buffer : Antibody Diluent, invitrogen,
Incubation time: 30 minutes wash Buffer: PBS Number of washes: two times
DETECTION METHOD HRP Detection
POSITIVE AND NEGATIVE CONTROLS USED -
ANTIBODY STORAGE CONDITIONS -20℃
FIXATION OF SAMPLE Yes/No : yes If yes: Fixative agent and concentration 10% neutral buffered formalin Fixation time : 24 hours Fixation temperature : room temperature
ANTIGEN RETRIEVAL pH 8.0 Tris-EDTA buffer & pH 6.0 citrate buffer & Proteinase K
PERMEABILIZATION STEP -
BLOCKING CONDITIONS not used Endogenous peroxidases blocked? 3% H2O2 room temperature 10 minutes Endogenous biotins blocked? Not used
SECONDARY ANTIBODY Pollnk-2 Plus HRP Detection kit (Golden Bridge International, Inc.) (Cat No D41-125) Species: Isotype: Reacts against: Mouse & Rabbit Concentration or dilution : Ready-to-use Diluent buffer Incubation time: 15 minutes Fluorochrome or enzyme conjugate: wash Buffer: PBS Number of washes: two times
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5
HAVE YOU RUN A "NO PRIMARY" CONTROL? No
DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
WHAT STEPS HAVE YOU ALTERED? Antibody dilution 200x and 50x Antigen retrieval: HIER(Citrate 6.0, EDTA 8.0), Enzyme(Proteinase K), No treatment endogenous peroxidase blocking time(6 minutes & 10 minutes).
ADDITIONAL NOTES -
Asked on Mar 07 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.
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 ab91007 is tested and covered by our 6 month guarantee for use inIHC-P andhuman 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 optimize the results from ab91007. I would also appreciate if you can confirm some further details:
1. Please confirm whichtype of tissuewas tested.
2. I can recommend to include 0.2% Tween in the wash buffer and antibody dilution buffer. Wash 4 times for 5 minutes at each wash step.
3. I suggest to try reducing the concentration of antibody which should help to reduce the background. Try 1:500.
4. It woudl be important to include a blocking step. For example, try 5% Serum for 30 minutes. Thismayrequire some optimization.
5. Is the secondary antibody working well with other primary antibodies? What were the results of the no primary control. The concentration may need to be reduced in order tooptimize the results.
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 information and details of how you would like to proceed.
Answered on Mar 07 2012