[Histonet] Some details about my questions on mouse olfactory bulb perfusion/fixation.

[Leyva-Grado, Victor]

Hi everybody,

 

I received some input about my questions on OB (thanks), but as some of
you noticed I didn't include in my previous mail some details in the
procedure that are important. So, here I go again.

 

After collecting the brain, we post-fix it in 4% PFA for 4 h at 4C and
then we sink the brain in 20% sucrose (usually overnight). After
sucrose, we froze the tissue with dry ice and then we store them at -80C
until we used them. We cut serial sections in a microtome (in a cold
platform, 30um) and then we split some sections for DAB staining and
some sections for double labeling.

For double labeling we tried adding the two Alexa fluorochromes at the
same time or using an incubation with Streptavidin Alexa 568 against the
first Ab and then Alexa 488 for the second antibody. In both cases we
had the endogenous fluorescence particularly in the glomerular layer,
which unfortunately is one of the regions of the OB that I'm most
interested in.

 

Thanks a lot,

 

Victor H Leyva

Washington State University