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Product code: 51075
Lot number: GR19885-6
Inquiry: Please see technical enquiry form attached.
Antibody Code: EP1270Y (ab51075)
Batch Number: GR19885-6
Antibody Storage Conditions (temperature/reconstitution etc)
Arrived in liquid form from manufacturer
Description of the Problem (high background, low signal, non-specific staining etc)
Sample (Species/Tissue/Cell Type/Cell Line etc)
Human placental tissue
Fixation of Sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration)
We have attempted on both formaldehyde and paraformaldehyde fixed tissue (duration of fixation is usually overnight at 4 degrees
Antigen Retrieval (Enzymatic method, Heat mediated technique etc)
We have attempted enzymatic (Trypsin, using Invitrogen’s Digest All Kit), and heating sections in citrate buffer as well as EDTA antigen retrieval buffer for 15-20minutes at high & low power.
We have tried Triton-X as well as
Blocking Conditions (Buffer/time period, Blocking agent)
We use Protein Block (DAKO) for 10minutes
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, wash step)
Abcam Antibody/Rabbit/1%BSA,PBS/1:10, 1:25, 1:50/1-2hr at RT or 37 degrees, overnight at 4 degrees attempted as well/washed with either PBS or PBST after primary antibody
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, wash step)
Invitrogen: Superpicture 3rd gen IHC kit/Universal including rabbit, mouse, rat/ready to use/10-30 minutes/wash with PBS
Positive and negative controls used (please specify)
Placental tissue is a positive control for MMP17. Placental tissue has been shown to express MMP17 by both Western Blot and Real-Time PCR methods.
OPTIMIZATION ATTEMPTS (PROBLEM SOLVING)
How many times have you tried the IHC?
30+ sections have been used to optimize the antibody, we have tried everything
Have you run a “No Primary” control? Yes (Delete one)
Do you obtain the same results every time? Yes (Delete one)
What steps have you altered?
Tried altering incubation time, antibody concentration, antigen retrieval methods (duration and solution)
The antibody does not work at the highest concentration recommended by Abcam (which was 1:50). We have tried even higher concentration without success.
Asked on May 23 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 provide us with vital information for our monitoring of product quality. I would like to reassure you that this antibody is tested and covered by our guarantee for IHC-P and human samples.
I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there not many tips to provide on this occasion to help improve the results. However, I can recommend you may like to consider trying the following options and considerations:
1. I can suggest to try less time for heat mediated antigen retrieval? This can sometimes require some optimization, and less time is sometimes better than more time. For example, try 5 minutes.
2. As MMP17 is a cell membrane protein, we would not recommend to permeabilize in Triton. Triton is a strong detergent which can disrupt membrane proteins. It can sometimes significantly affect the staining of membrane targets.
3. Is the IHC kit working well with other primary antibodies?
Alternatively, or if these tips do not work, I am pleased to offer you a free of charge replacement or credit note in compensation if the item has been purchased in the 6 month guarantee period. In this case I would appreciate if you are able to confirm the order number.
Thank you for your cooperation. I look forward to hearing from you with the results of the suggestions details of how you would like to proceed.
Answered on May 23 2012