fixation of frozen sections for H&E versus immunostaining

From:Gayle Callis

Morphology is alway compromised by certain types of fixation for frozen

Our frozen sections destined for H&E stain, are cut and immediately placed
into NBF at RT in order to have the best morphology.  They can fix for
several minutes, a minimum of 1 min, or sit in NBF for weeks before staining. 

We found 95% ethanol to be very dessicating, the tissues show poorer
morphology and acetone is reserved for immunostaining only, it was the
WORST for an H&E. 

Immunostaining usually means a panel of fixtions to optimize for staining,
and we HAVE found differences with some antibodies as to how FS fixation is
done. One antibody in particular was horrible after acetone, but excellent
after acetone/alcohol method.  

1.  For our murine research IHC, we fix with 4C acetone, single fixation
for 10 min after air drying the sections for a min of 30 min in front of a
small fan to overnight over 16 mesh silica gel.

2.  Air dry sections  min of 30 min to overnight, same as above, a double
acetone fixation method that improves morphology but keeps FS on slides

Acetone 4C 10  min
Air dry 10 - 15 min in front of a small fan
Acetone 4c 10 min
Air dry 20 min, proceed to IHC

3.  A favorite is acetone 75%/absolute ethanol 25%.  Air dry sections
overnight, fix in AA for 5 min at RT, go directly to 3 changes of buffer
(DO NOT AIR DRY AFTER THIS FIXATIVE).  This works well for our murine cell
surface markers, but may not work well for human CD4 or CD8.  The
morphology is superior to just acetone. 

I have done unfixed frozen sections for immunostaining, but fixation was
done AFTER primary antibody incubation. This is dicey with extremely
delicate FS, must be rinsed very CAREFULLY.  Incubation of antibody was
carried out, flat in the refrigerator. 

There are other fixatives to try IHC.


Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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