I am trying to use HIF-1 to identify hypoxia in sections of 3D neuronal cultures. I have PFA fixed cells and sections and I cannot make any antibody work. I know for sure I have hypoxia as the Hypoxyprobe is positive but cannot make any HIF-1 Ab work consistently and I would like to co-stain for vulnerable cell populations. Any ideas on a protocol that might work? I currently have the HIF-1 Ab [H1alpha67] - ChIP Grade (ab1) from Abcam. Thank you in advance!
Use DMOG to induce HIF as a positive control. The Cell Signalling HIF antibody works well.
the protocol is tricky and -in our hands- only works with a tyramide-based amplification step. antigen retrieval: 5 minutes in pressure cooker with DAKO target retrieval solution; primary antibody: Cayman 100006421 (works on murine and human sections!) 1:20.000 (no typo!), then proceed with DAKO CSA Kit II (or similar tyramide-based amplification, e.g. from Perkin-Elmer). inlcude a positive control, e.g. normal colon or small intestine (HIF-1a stabilization under normal conditions in luminal enterocytes due to physiologic oxygen gradient). keep in mind that hypoxyprobe-positive areas not always co-stain for HIF-1a.
good luck!
Thorsten
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