[Histonet] autofluorescence?

From:yasushi nakagawa


We are doing immunohistochemistry using Cy2, Cy3 and Cy5-conjugated  
secondary antibodies on 20um-thick cryosections of embryonic mouse  
brains. Especially with Cy2- filter setting, we sometimes see a  
number of auto-fluorescence-like signals within the brain sections.  
They are thin and long, kind of looking like blood vessels. They show  
up even when we do not use Cy2-conjugated secondaries. The same  
problem has happened to Cy3-filter setting, too, although less  
frequently. I wonder if anyone has a idea about what they are and how  
to prevent them from showing up.

We fix brains in 4%PFA/PB overnight, wash and cryoprotect in sucrose/ 
PB, and freeze in OCT in plastic molds on petri dish on liquid  
nitrogen. We cut 20um cryosections, dry them for an hour or so, and  
do regular antibody staining, do DAPI staining, dehydrate, and mount  
in DPX.

Thank you for your help.

Yasushi Nakagawa

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