Reply: alternative fixation/processing schedules for sensitive antigens

From:Gayle Callis

Yes, and it works, you can do paraffin sections - they tend to be drier,
more brittle, require some ice water soaking after trimming block. You have
to work with Alk Phos substrates, you cannot quench endogenous peroxidase -
had a friend who tried really concentrated hydrogen peroxide mixtures
without success.  He should publish these results. 

The concentrations for murine antibodies is almost the same as for acetone
fixed frozens AND you never have to do antigen retrieval. 

I recommend a time study for optimal fixation, fix only small tissues, not
big slabs or you will end up finishing off fixation during processing - not
what you want to do.   

Tissue processing used just routine alcohols and xylene, into your favorite
paraffin.  We did shorten processing times for mouse.

There are other fixative combinations for frozens with poor morphology
using acetone. I frequently use, and found one antibody worked best
(acetone shot it down!) with acetone (75 mls)/absolute ethanol (25ml) after
overnight air drying, fix in this fixative for 5 min at RT, go to PBS
rinses X 3 immediately, do not air dry again, proceed with staining.  I do
use the glucose oxidase peroxidase blocking method for
frozens/immunoperoxidase staining. 

My murine CD4 (L3T4) clone has been worked up with a dilution of 1:15000 -
20,000 (0.5mg/ml, Pharmingen) using DAKO DAB+ and their DAB enhancer on
frozens fixed in acetone/alcohol mixture.  Fun to tweak the system.  

I give credit to Barb Wright for putting me onto this fixation method.

There are some original publications on this fixation method original was
on human, other was on murine cell surface markers.  
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)

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