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Hello, |
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ANSWER: |
Thank you for contacting Abcam. |
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DESCRIPTION OF THE PROBLEM No staining. Just hazy in general SAMPLE Adult mouse brain PRIMARY ANTIBODY overnight at 4 degrees in MBP antibody 1:1000, and 1:500 DETECTION METHOD fluorescence microscope POSITIVE AND NEGATIVE CONTROLS USED adult brain ANTIBODY STORAGE CONDITIONS At time of staining, had just reconstituted. Currently at 4 degrees FIXATION OF SAMPLE perfused with paraformaldehyde ANTIGEN RETRIEVAL Sections were double stained for BrdU so sections were first incubated in 2N HCl for 30 min at 37 degrees then immediately in borate buffer for 15 min at room temp. After 3 washes, sections were incubated in .3% hydrogen peroxide for 10 min. PERMEABILIZATION STEP see above BLOCKING CONDITIONS 1 hr room temp in goat serum SECONDARY ANTIBODY 1 hr at room temp in alexafluor 488 1:1000 HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? concentration of primary--1:1000 as recommended and 1:500 ADDITIONAL NOTES Have used protocol and secondary many times. |
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ANSWER: |
I'm sorry to hear you are experiencing problems with ab2404. I think the problem might be due to the following: -the antibody may not recognise mouse MBP. The homology of the immunogen with mouse protein was found to only be 71%. It would be useful to therefore try a positive control of human or rat samples - the pretreatment of tissue with HCl may damage the epitope and prevent the antibody from recognizing the protein. I therefore recommend to try without the HCl (and borate) pretreatment. -the perfusion method may not be adequate. The antibody has been tested on PFA-picric acid perfused tissue. The presence of picric acid may be key to a good staining. You do not mention how long you postfix for, this may also affect the epitopes and prevent good recognition by the antibody if the tissue is fixed more than by perfusion. I would finally recommend to add in the blocking buffer and antibody buffer 0.3%v/v triton X100 to help the penetration of the antibody in the tissue. I hope these recommendations will help you, |
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I would like to stain myelin basic protein in paraffin-embedded brain tissue using chromagen stain. Would this antibody work for this?
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ANSWER: |
Thank you for your enquiry. Yes, Abcam's ab2404 Myelin Basic Protein antiserum can be applied using immunohistochemistry. We suggest antigen retrieval followed by an incubation period of 30 minutes at room temperature using a dilution range of 1/50 - 1/100 (ABC method). I hope this information helps. Please do not hesitate to contact me should you require any further assistance. |
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