[Histonet] Autofluorescence using Mouse Tibia Tissue

Branden Smeester smees001 <@t> umn.edu
Wed Jan 19 15:32:18 CST 2011

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


block (2-10% normal serum in TPBS)
primary overnight
PBS washes
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.


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