[Histonet] Re: Bone Frozen sections versus decalcified bone in paraffin

[gayle callis]

Louise, 

 

You wrote:  last year sometime I was asked to budget for some equipment for
our unit.Not expecting to get anything i aimed high, and requested a
cryostat and cryoJane system.

Lo and behold, the planetary influences were just right, and my request was
approved. Now, I've got cold feet (pardon the pun) and I'm wondering if
there *are*  any distinct advantages of cryosections of bone over

demineralised wax embedded samples in regards to immuno and in-situ ?.

 

Yes there are distinct advantages for using Cryojane.   Undecalcifed bone
frozen sections are done in our lab in order to do immunostaining, either
enzyme immunohistochemistry or immunofluorescence work on fresh undecalcifed
bone frozen sections, especially when murine CD markers and GFP are
compromised by aldehyde fixatives (PFA or NBF), acid decalcification, or
aldehyde induced autofluorescence when working with GFP .  There are ways to
get around the autofluorescence  problem.  We use Cryojane for all
undecalcifed bone frozen sections, but also section other very difficult
frozen tissues.  There are times using FFPE/decalcified bone is not going to
stain successfully for either immuno or ISH methods IF the antigens are
compromised by either mentioned methods, plus heat from paraffin processing.
Cryojane is our backup method when things go awry, and our method of choice
with most GFP work with murine nasal turbinates.    Trying to do "free hand"
frozen sections on undecalcified bone was an unsuccessful nightmare, very
time consuming with terrible results (crunched up bone sections).    Our
problem has been too many murine CD markers we work with only stain on fresh
frozen sections after solvent fixation.  If most of your antigens (or ISH)
stain successfully with decalcified, FFPE sections, then you may not need
Cryojane but that is something you have to work out.  We find Cryojane a
necessity for our work.    

 

One can decalcify bone prior to fixation Mori et al then cut frozen
sections, fix and stain - a long drawn out procedure for IHC.  There are
others who have success on undecalcifed bone frozen sections ( Kusser et al
with excellent references)  Both of these publications are now freely
accessible in  J Histochem Cytochem.  ISH is more difficult on acid
decalcified bone, where EDTA may have to be used for FFPE.  There are some
excellent publications on using decalcified bone (marrow) with ISH, the most
recent from Gudrun Lang in Journal of Histotechnology, March 2010 describing
kappa and lambda ISH on acid bone marrow biopsies.   She listed several
excellent references on ISH/IHC for FFPE bone samples.   Gudrun participates
on Histonet and should be able to drop a pdf of this publication to you.  I
have also done PLP fixed, EDTA decalcified bone, sucrose cryoprotected bone
frozen sections mounted on Plus Charge slides, but prefixed bone frozen
sections often release from the slide surface during staining. That is the
joy of Cryojane, no release.  Working with the instrument takes some
practice, and there is an excellent publication by John Tarpley in J of
Histotechnology for doing this.  

 

Cryojane is unique but be sure it fits in your cryostat before purchase.
There is the Kawamoto tape method for bone frozen sections, but it requires
purchasing a knife holder, and I believe,  special knives.  The section is
NOT transferred to a slide, but remains on the tape where all staining takes
place, then mount the tape onto a slide in a special way.  Those who use it
acquire beautiful staining results for both IHC and ISH.  This method has
been around for awhile and you have to purchase all the accessories and
tapes from a company in Japan.  I know that Jamie Erickson is using this and
has also worked with Cryojane.   Hopefully Jamie is looking in and can
comment.  

 

Good luck, 

 

Gayle M. Callis

HTL/HT/MT(ASCP)