[Histonet] Questions about the mouse olfactory bulb

[Leyva-Grado, Victor]

Hi everybody,
 
I'm working with mouse olfactory bulb (OB) for DAB-IHC and Alexa fluor
double labeling. I've found several difficulties and I'd like to have
your input about them.
 
We perfused the mice with 4% PFA using a syringe (first saline then PFA)
and we found out that usually the brain is well perfused and fixed, but
the OB not always gets well fixed. Any suggestions on how can we improve
the OB fixation to conserve better the histology of the tissue? A bad
fixation can result in unspecific DAB labeling?
 
In the other hand, when we do the immunofluorescence, the region of the
glomerular layer is always fill with unspecific dots of fluorescence. Is
this "endogenous fluorescence" or is a consequence of bad
perfusion/fixation?
 
Thanks a lot for your comments,
 
Victor Leyva
Washington State University