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BATCH NUMBER lot 8392 DESCRIPTION OF THE PROBLEM No Signal. SAMPLE human buccal mucosa PRIMARY ANTIBODY ab 11217 anti keratin-guinea pig polyclonal to keratin. Diluted 1:20 with TBS and 1% BSA. Incubated at 4oC for ~18hr in a humidified box. Slides washed 3x5mins in coplin jars containing TBS. SECONDARY ANTIBODY ab6567 Anti-IgG, Goat polyclonal to guinea pig IgG h&L (Cy5). Diluted 1:250 in TBS with 1% BSA. Incubated for 60 mins at room temp in a humidified box. Slides washed three times in TBS for 5 mins each. DETECTION METHOD Secondary contains cy5, slides stained lightly with DAPI and viewed under fluorescent microscope. Microscope has triple band filter for simulataneous viewing of DAPI and Cy5. POSITIVE AND NEGATIVE CONTROLS USED Negative control buccal cells with no primary anti body. Could you suggest an appropriate posative control compatible with your antibody.Some cell lines are keratin sub group specific. ANTIBODY STORAGE CONDITIONS Stored in fridge at 4oC, not frozen FIXATION OF SAMPLE sample air dried for 10mins and then fixed in ice cold acetone for 10 mins. ANTIGEN RETRIEVAL Did not include this step as using cell suspension on a slide and not paraffin block. PERMEABILIZATION STEP By fixing in ice cold acetone does this aids in permeabilising cell membrane? BLOCKING CONDITIONS 20% goat serum in TBS for 30 mins at room temperature in a humidified box. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Blocking step for longer, and triton post hybridisation washes. ADDITIONAL NOTES i am primarily using buccal cells for immunohistochemistry experiments.
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ANSWER: |
Thank you for your enquiry and for taking your time to fill in the on-line Questionnaire. Keratin is expressed in the terminally differentiated epidermis of palms and soles. We would suggest using human or cow skin as positive control but please be aware that this antibody does not react with mesenchymal, glial, neuronal or muscle cells. We could recommend using ethanol or methanol but ice-cold acetone (5 or 10 min fixation) should really work. You are right, acetone will fix and permeabilise the cells so you do not need to apply a permeabilization step. Try to fix the samples as soon as possible and do not wait until they are completely dry. Please do let us know how you are getting on. Should you still have problem with this antibody, then please do not hesitate to contact us again.
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