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Please find the answers to the questionnaire.
Images are attached.
I still hope that the technical teams are able to provide me solutions,
otherwise I'd like to have refund for this product.
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1) Abcam product code ab 47308
2) Abcam order reference number 1081728 or product batch number
3) Description of the problem false positive staining
4) Sample preparation: pancreas
Type of sample: Fresh frozen sections, perfusion fixed frozen sections,
PFA/formalin fixed paraffin embedded sections, cells in culture, other:
Positive control N/T
Negative control normal IgG
5) Fixation step
If yes: Fixative agent and concentration 4% Paraformaldehyde (for frozen
Fixation time 15min
Fixation temperature room temperature
6) Antigen retrieval method for FFPE sections, xylene dewax, followed by
either sodium citrate buffer (pH6.0) for 10min or 25min / Tris-EDTA (pH9.0)
for 20min / EDTA (pH8.0) for 20min in scientific microwave
7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing
agent in any dilution buffers? Yes
Permeabilizing agent and concentration: Two methods were tested
1st method: diluting primary antibodies in dilution buffer (1xPBS, 0.1%
Triton-X100) and incubate for 2hrs at R.T.
2nd method: before incubating with primary antibody, sections were
permeabilized using 1xPBS buffer, 2% Triton X, 0.2% Tween20 for 1hr.
8) Blocking agent (eg BSA, serum…): normal goat serum as secondary
antibody was anti-guinea pig raised in goat
Blocking time blocked simultaneously with primary antibody incubation
Blocking temperature room temperature
9) Endogenous peroxidases blocked?
Endogenous biotins blocked?
10) Primary antibody (If more than one was used, describe in "additional
Concentration or dilution 1:50
Diluent buffer 1xPBS, 0.1% TritonX-100
Incubation time 2hrs at r.t.
11) Secondary antibody: Fluorescent Texas Red
Reacts against: guinea pig IgG
Concentration or dilution 1:200
Diluent buffer 1xPBS, 0.1% TritonX-100
Incubation time 1hr
Fluorochrome or enzyme conjugate Texas Red
12) Washing after primary and secondary antibodies:
Number of washes 3times
13) Detection method fluorescent microscope 40x magnification
14) How many times have you run this staining? 3
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody?
Antigen retrieval buffer, permeabilization
Document attachment: Attaching images of your IHC is strongly recommended
and can greatly speed up our investigation of your problem.
Asked on May 30 2012
Thank you for taking time to complete our questionnaire. I am sorry to hear that 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.
Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab47308 Anti-PDX1 antibody:
1. As you are performing a heat- induced antigen retrieval, an extra permeabilization step is not necessary for the FFPE.
2. Triton can be quite harsh on some epitopes, therefore, you may increase the clarity of the staining by reducing the Triton concentration during . Still, I can recommend keeping the Triton to your incubation solution to facilitate the antibodies solubility, but in a concentration of 0.025%.
3. As the necessary permeabilization step for the frozen sections, I would like to suggest to decrease the permeabilization step time with 0.1% Triton-X100in PBS down to 30 min.
4. To assure that the non-specific binding site are blocked, please block with at least 5% serum for 30 min before the antibody incubation.
5. The antibody concentration is very high compared to what we suggest on the datasheet, especially as the used tissue is the positive control. I would therefore suggest to increase the specificity of the antibody by decreasing the concentration down to at least 1:500 and to incubate overnight at 4C.
Hopefully, this will bring down the non-specific binding. Should the suggestions not improve the results, please do let me know.
In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.
I hope this information is helpful, and I thank you for your cooperation.
Answered on May 30 2012