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LOT NUMBER gr9066-6 ORDER NUMBER 984557 DESCRIPTION OF THE PROBLEM Non-specific staining SAMPLE Species: mouse What’s cell line or tissue: skin PRIMARY ANTIBODY Species:rabbit polyclonal Reacts against: human mouse cow dog At what dilution(s) have you tested this antibody:1:50/1:500 Diluent buffer: DAKO antibody diluents buffer (ready to use) Incubation time: overnight Incubation temperature: +4℃ What washing steps were done (which buffer, number of washes): 1x PBS/3 times/5 mins DETECTION METHOD AEC ANTIBODY STORAGE CONDITIONS -20℃ FIXATION OF SAMPLE Type of sample (Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed ˙paraffin embedded sections, cells in culture, other:____) : PFA fixed paraffin embedded sections ANTIGEN RETRIEVAL Antigen retrieval method(including time, temperature etc.): 1X Tris-EDTA PH9 Time:15mins/10mins Temperature:99~100℃ PERMEABILIZATION STEP Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers?No ˙Permeabilizing agent and concentration: BLOCKING CONDITIONS Blocking agent (eg BSA, serum…): superblocking buffer (pierce 37515) Concentration:ready to use Blocking time :1hr Blocking temperature: room temperature Endogenous peroxidases blocked? 3% H2O2 methanol room temperature 10 mins SECONDARY ANTIBODY Species:DAKO LSAB2 System Reacts against which species: mouse/rabbit/goat At what dilution(s) have you tested this antibody: Diluent buffer: Incubation time:biotinylated secondary antibody and streptavidin-HRP reagent 30mins/each What washing steps were done (which buffer, number of washes): 1x PBS/3 times/5 mins Fluorochrome or enzyme conjugate (eg: FITC, HRP, AP, biotin…etc): biotinylated secondary antibody and streptavidin-HRP reagent Signal amplification method (eg: ABC, LSAB, HRP polymer, TSE): LSAB HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? 1.Primary antibody diluent ratio 1:50/1:500 2.Antigen retrieval time 15mins/10mins
Asked on Dec 05 2011
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 ab21607. I would also appreciate if you can confirm some further details: In many cases, antigenicity of elastic fiber components can be enhanced by treating with 6 M guanidine HCl, 50 mM dithiothreitol, 20 mM Tris pH 8.0 for 15 minutes. After washing with 20 mM Tris pH 8.0, the slides are treated with 100 mM iodoacetamide in the dark for 15 minutes, washed and stained using normal protocols. I would try including this step. A number of other researchers on IHC world have stained Elastin successfully be using proteinase K antigen retrieval at 37°C for 20 min. This is another step I would try but without the pretreatment detailed above. How was the antibody treated prior to storage at -20°C? Glycerol should be added to a final volume of 50% prior to storage. 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 Dec 05 2011