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DESCRIPTION OF THE PROBLEM Non-specific staining SAMPLE Human Paraffin Embedded sections PRIMARY ANTIBODY Concentration or dilution : 1:50, 1:100, 1:200 Incubation time : 30min at RT DETECTION METHOD - POSITIVE AND NEGATIVE CONTROLS USED - ANTIBODY STORAGE CONDITIONS -20C FIXATION OF SAMPLE 10% formalin ANTIGEN RETRIEVAL Enzyme: proteinase K HIER: EDTA 8.0: EDTA buffer pH 8.0(PT Module Buffer 3, TA-125-PM3X) EDTA 9.0: EDTA buffer pH 9.0 (PT Module Buffer 4, TA-250-PM4X) Citrate: Citrate buffer pH 6.0 (PT Module Buffer 1, TA-125-PM1X) PERMEABILIZATION STEP - BLOCKING CONDITIONS Concentration : Ultra V block (Thermo (구 Labvision사) 제품 #TA-060-UB) Blocking time : 10min Blocking temperature : RT Endogenous biotins blocked? 0.3% H2O2 SECONDARY ANTIBODY Secondary antibody: Thermo #TL-060-HD Reacts against: anti mouse igG and anti rabbit IgG Incubation time : 30min Fluorochrome or enzyme conjugate HRP HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? - ADDITIONAL NOTES Folia Histochemica Et Cytobiolocia 48: 475 ~ 483, 2010 Nerve tissue expresses CGRP, Substance P but, this antibody stained all tissue non-specifically.
Asked on Dec 21 2011
Thank you and your customer for taking the time to complete our questionnaire and contact us. I am sorry to hear your customer has had difficulty obtaining satisfactory results from this antibody. The details your customer has kindly provided will enable us to investigate this case for your customer and this is also helpful in our records for monitoring of quality. Reviewing this case, I would like to offer some suggestions to help optimise the results from ab47027: 1.) To reduce un-specific staining I suggest to increase the blocking time to 1 hour. 2.) For the most specific staining and also a full saturation of the tissue with the primary antibody, I strongly recommend that the incubation time is increased to over night at 4C. 3.) To determine that the background staining is not due to the secondary antibody, I suggest to run a no primary control. 4.) I also recommend to make sure that the samples does not dry out since these samples than produce high background. I would also appreciate if your customer can confirm some further details: 1.) Unfortunately, I received the wrong image (WB and different antibody). Could you please provide an image of the IHC-P experiment? 2.) What sample/ tissue was used for this experiment? 3.) It is stated that the experiment was repeated three times- which antigen retrieval methods were used and what were the timings of them? 4.) For how long was the sample fixed in 10% Formalin? We are happy to offer this technical support. In the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund. I hope this information is helpful, thank you and your customer for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.
Answered on Dec 21 2011