[Histonet] questions about immunofluorescence on frozen tissue
I have the following questions: Iím trying to do immunofluorescence on frozen tissue. I had tried both 4% formaldehyde and AA fixations. It seems that 4% formaldehyde give the better morphology but weaker signal than AA fixation. I wonder whether it is necessary to apply the permeabilization step in a frozen tissue section. I had saw two different opinions about this. Some people said we donít need to permeabilztion, since the cell interior is fully exposed to antibody in the cutting sections. Some said there is need to add this step to get better results. In previous, Chris van der Loos mentioned that ďAt the end of the IHC staining procedure, after the chromogen step wash your slides with tap water. NEVER use distilled water as this will ruin the tissue section completelyĒ. We also conterstain DNA with DAPI. My question is that should we wash slides with tap water after 2nd antibody-conjugated with chromogen or after conterstaining with DAPI? Also Chris mentioned
about avoiding using tween-20 in AA fixed slides. If elimiating tween-20 from the washing buffer, I encountered a bubble problem while putting the coverglass. There is no or weaker signal from those regions cover by the bubbles. Can anybody teach me how to avoid creating bubble while applying the coverglass? The mounting medium was bought from Vector. Any help will be highly appreciated.
Department of Epidemiology
School of Public Heath
University of California, Los Angeles
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