[Histonet] In situ background on fresh frozen sections

[yasushi nakagawa]

Hi,

We are doing in situ using fresh frozen adult brain sections. We find  
some sense probes have strong labeling in areas with high cell  
density, such as dentate gyrus, cerebellum and rostral migratory  
stream. I wonder if any of you know specific solutions to get rid of  
this problem. Does RNase treatment help? Is it compatible with the  
following protocol? We use similar protocols for embryonic and early  
postnatal brain sections (except that we fix them before freezing),  
and work great.

Our protocol:
Freeze fresh brain in OCT on petri dish on liquid nitrogen
Cut 20um cryosections
Dry for a few days
Fix 10 min in 4% PFA
Wash in PBS
Acetylation
Wash in Triton X-100/PBS
Wash in PBS
Prehybridize for 1-2h (based on Denhart, salmon sperm DNA, yeast RNA,  
50% formamide, SSC)
Hybridize with ~100ng/ml DIG-labeled probes at 72 degree overnight  
(~14hr)
Wash in 0.2xSSC for 1h at 72 degree
Change to B1 solution
Block for an hour (10% lamb serum in B1)
Antibody incubation with anti-DIG (Roche, 1:5000) in the cold room  
overnight
Wash in B1
Change buffer to B3
Do color reaction in NBT/BCIP with levamisole

(B1 and B3 are Tris-based buffers used in Roche protocols)

Thank you for the information.

Yasushi