[Histonet] questions about immunofluorescence on frozen tissue

From:Kelly Yang

      Hi Histolanders, 
  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. 

Kelly Yang
Graduate student 
Department of Epidemiology
School of Public Heath
University of California, Los Angeles
ext. 57795
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