[Histonet] Oil red O on GMA sections

[Gayle Callis]

Dear all

This is NOT an oil red O for undecalcified bone in GMA, but a general Oil 
red O for lipids in soft tissues that are NBF fixed.

Technovit 7100 is a kit and I have not used it but I have cut tissues 
embedded in this GMA, it is superb and a bit pricey.  Personally, I do not 
do like GMA embedded undecalcified bone, it has many inherent problems one 
of which can be problems with controlling polymerization along with poor 
infiltration plus being difficult to section.  GMA is just plain SOFTER 
than MMA!

Nancy, if I were to embark on a project like this, I would prefer an osmium 
tetroxide post fixation, then embed into MMA and section.  The lipid will 
be black, and remains in the tissue during processing and embedding as it 
does with epoxy resins for EM.

First, with any GMA for lipid, you cannot do any dehydration in alcohols, 
you would have to do a monomer gradient (expensive with a kit like 
Technovit 7100, archives will say where that can be purchased from) using 
decreasing amounts of water.   Sharon van de Velde simple uesd Monomer A 
from the JB4 kit, PolySciences or the monomer from the Technovits kit.  You 
need to do extra changes to remove all the water. Using the monomer from a 
Technovits kit could be very expensive unless you buy extra for this 
special processing.

Remember that GMA polymerization is very difficult to control in a thick 
bone sample, you may be limited to the usual 1 -2 mm thickness with 
cortical bone, although it works well with Jamshidi needle biopsies.

The oil red O protocol is the standard Oil red O found in histology text 
books, using propylene glycol.  Unfortunately, I do not have this on 
computer file but I do have the Churukian oil red o, I will attach that to 
you personally.   You may have to look it up or it should be available in 
Histonet archives, many people have responded with their recipes in 
past.   I know the standard propylene glycol method, how to make up stain 
is found in some website links found on Histosearch website.

Staining is for GMA sections (2 um thick!) with standard Oil red O in 
propylene glycol, a messy method!

absolute propylene glycol for 2 min
oil red O for 2 hours
differentiate in 85% propylene glycol 1 - 2 min
Hematoxylin counterstain
Mount with aqueous mounting media

I suggest you try the Churukian Oil red O stain, much nicer than messy 
propylene glycol but you would have to work out staining time.


Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)