Re: [Histonet] fluorescent labeling of fixed, frozen tissue

From:"Gayle Callis"

Many people do immunfluorescent staining on NBF perfused tissues but yours 
has unique problems involved.  Bone, decalcification and then cryosections 
of boney parts.

Unfortunately, fixed/decalcified bone frozen sections do not like staying on 
slides, even after air drying, which will make retrieval difficult too. 
You should not have to acetone fix after perfusion fixation, since the 
proteins are already cross linked, the acetone is probably not going to help 
that much with section retention.  If you do fresh tissue, decalcified and 
then fixed with acetone, the section should stay on the slide surface.   Air 
drying is one way to help, but may a gentle heat at 37C can help 
particularly if you use a protein coating, e.g. poly l lysine or possibly 
gelatin subbed slide to act as the glue for the section to slide surface. 
This was discussed on Histonet not too long ago.

See cited references below.  People who have used gelatin subbed slides can 
give more insight on its use for immunofluorescent staining.

Is there any reason you can't perfuse fix, then decalcify and do paraffin 
embedded sections of the cochlea?   The autofluorescence level will probably 
be the same as FF/EDTA decalcified/frozen sections anyway or is the antibody 
not going to work on FFPE/decalcified tissue.   It seems to me if the 
antibody works on FF/decalcified frozen sections (section fall off slide), 
then it should work on FFPE (also decalcified) that stay on slide better, 
they why not do the latter and not cryosection?  This way you can dry the 
slides longer to ensure section retention is improved although retrieval may 
have to be low temperature instead of harsher HIER methods.

You probably are suffering from autofluorescence induced by the formaldehyde 
fixation.  Formalin isn't fluorescing, it is inducing the autofluorescence. 
This will happen on tissues NBF fixed/decalcified for paraffin sections or 
for frozen sections.  To read up on this, go to IHCworld website.  You might 
be able reduce autofluorescence with 100 mM glycine in TRIS buffer, pH 7.4 
(we also use Dulbeccos PBS at the same pH with 100 mM glycine.  You 
rehydrate a paraffin section (frozen sections can be flooded with the 
solution) for 20 min at RT before proceeding to immunfluorescent 
immunostaining or apply it to a fixed frozen section.

The other alternative although very pricey, is use Instrumedics Cryojane and 
then you can tape transfer any frozen section of bone, whether it is fixed 
or not to a slide surface.  Unfixed, acetone fixed bone frozen sections 
should have minimal autofluorescence.  Some people complain about the 
polymer on the slide but there are ways to deal with that problem.

There is an excellent publication on doing decalcification/cryotomy for 
eGFP, and they may also do immunostaining too.  Kusser and Randall,  J 
Histochem Cytochem 51:5-14, Jan 2003.

You can also decalcify unfixed bone, a long tedious protocol, snap freeze 
and then fix with acetone after the EDTA decalcification is complete.  You 
will NOT be able to do microwave decalcification. this is done at 4C.
      Mori S et al.   A new decalcifying technique for immunohistochemical 
studies of calcified tissue, especially applicable to cell surface marker 
demonstration. J.       Histochem Cytochem. 36(1):111-4,1988.

As for an easier way, we looked for them too, and ended up buying the 

Good luck

Gayle M. Callis
Bozeman MT 59715

----- Original Message ----- 
From: "Bower, Jennifer" 
Sent: Thursday, April 17, 2008 3:32 PM
Subject: [Histonet] fluorescent labeling of fixed, frozen tissue

Hey everyone!

   I have a big long question, I hope someone out there can help me.
Ultimately, I need to do fluorescent labeling of formalin fixed,
decalcified mouse cochlea. Right now, we are perfusion fixing mice with
10% NBF, dissecting out the bulla with the cochlea still within,
decalcifying with EDTA in the microwave, then freezing the samples and
cutting sections on the cryostat. So far, I have to let my slides air
dry because if I put them straight into PBS, the sections fall off the
slides. Also, everything fluoresces, I suspect due to autofluorescence,
or maybe because of the formaldehyde? Isn't there an easier way? If the
tissue is already fixed, why put the sections into acetone after
cryosectioning? Do I need to do antigen retrieval? Should I try a
pressure cooker method? There is also a lot of superstition surrounding
the use of fluorescents with paraffin embedded tissue, no one seems to
think it will work. Is anyone out there doing any sort of fluorescent
labeling of fixed tissues? Thank you so much for any advice, I feel lost
at this point.

Thanks again,


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