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DESCRIPTION OF THE PROBLEM No staining or low staining although I know there should be at lot in this tissue. I see approximately one distinct dot in each cell if I am lucky with the staining. How is it supposed to look? Can you send me a picture of an immunohistochemistry staining? SAMPLE sections of rat brain tissue, looking at hippocampus PRIMARY ANTIBODY GR anti rabbit, over night 4C, different concentrations ANTIBODY STORAGE CONDITIONS -20C FIXATION OF SAMPLE 4% paraformaldehyde over night at 4C ANTIGEN RETRIEVAL sodium citrate, pH9, at 80C for 30 minutes, or no pretreatment BLOCKING CONDITIONS blocking with donkey serum 3%, 30 min SECONDARY ANTIBODY donkey antirabbit Alexa 488 (Molecular Probes) always works in all kinds of stainings HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No WHAT STEPS HAVE YOU ALTERED? concentration, pretreatment, incubation time
Asked on Apr 19 2006
Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. Rabbit polyclonal to Glucocorticoid Receptor (ab3579) is a good selling antibody. It is yet to be tested by IHC using paraffin embedded sections but has been shown to work using frozen sections. I find the fact that you are observing a distinct focus strange given that this is a cytoplasmic protein that becomes nuclear following the binding of its ligand. I would like to recommend that you fix your rat brain tissue in a mild fixative such as a 5 minute incubation of ice cold acetone or alternatively methanol. This approach does not carry the potential epitope modification caused by paraformaldehyde or formalin and does not require antigen retrieval. Can you please confirm that you have been using a secondary antibody against rabbit IgG as this was not detailed in your questionaire. Furthermore you have not provided details of the dilution. I would recommend a dilution series encompassing 1/100 to 1/1000 I hope this information helps, please do not hesitate to contact us if you need any more advice or information.
Answered on Apr 21 2006