[Histonet] Rinsing Michels transport media from skin biopsies

From:Gayle Callis

We always used the Citrate Stock/Rinse buffer made specifically for Michels 
transport media

Stock Citrate Buffer
1M potassium citrate buffer pH 7  2.5 ml
0.1M Magnesium sulfate   5 ml
  0.1M N-ethyl malemide    5 ml
distilled water 87.5 ml

Working solution i.e. Michels transport medium is made up in the same 
buffer with a salt added

ammonium sulfate   55 g
Stock buffer            100 ml

The ammonium sulfate precipitates the immunoglobulins you want to stain, 
and this precipitation is reversed by rinsing the tissue with the Stock 
buffer at least 3 changes and with larger pieces of tissue, rinse longer - 
we did 3 changes/10 minutes each change - the suggested agitation is a good 
idea, on a rocker or with stirring, then blot and preferrably snap freeze 
to prevent excessive freezing artifact.

Go to this website:   www.zeusscientific.com to read up on this fixative 
and how it is used.  We much preferred to buy the buffer ready made as it 
was a pain to make up all the time.

Barry Rittman made an excellent suggestion is to embed in the OCT (Sakura 
Finetek) and let sit for a short time before snap freezing.  This sometimes 
gives a better interface of cryoembedding media and the tissue.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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