[Histonet] Autofluorescence using Mouse Tibia Tissue

[Branden Smeester]

Hello Histonetters,

Currently, I am having trouble achieving good immunohistochemistry results
using frozen mouse tibia sections. I use the protocol as follows:

4% PFA or 10% NBF 12-24 hrs
cryoprotect in 20% sucrose until tissue sinks
freeze tissue in block molds using OCT compound and isopentane/dry ice
slice on cryostat (anywhere from 5-100um depending on microscopy work being
completed)

IHC:

block (2-10% normal serum in TPBS)
primary overnight
PBS washes
secondary
PBS washes
fluorescent mounting media

I am using Cy3 labeled antibodies and not achieving that good staining I
would like. Does anybody know a protocol or some special tricks I can use to
avoid the autofluorescence? I can't seem to have any luck since slicing
mouse bone tissue. I know many people have problems, but many publications
have achieved good results.

Thanks,
Branden