F.S. Ganglia identification

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From:BB racing <bbracing@silk.net>
To:Histonet <histonet@pathology.swmed.edu>
Date:(No, or invalid, date.)
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Linda: re your problem of ganglia identification in F.S biopsies
The following is a method that I worked out in the early 70's when for some unknow reason we had a dozen or so babies born, over a short one year period, with, or suspected of having Hischsprungs disease.  The method is very simple and very rapid, and should solve your problem.  It is based on the fact that ganglia are quite rich in the dehydrogenase enzyme, which is quite easy to detect, and stands out very clearly in the frozen section material.

1.  Cut your frozen section at between 6U to 10U (the thick sections make it easier to detect the      ganglia if they are sparsely located in the biopsy)
2.  Pick your sections up on a slide (charge slides work best) and air dry for one minute.
3.  Pipet a few drops of substrate directly onto the slides at room temp for 10 min. ( I usually cut      two slides and remove one after 5 min, and it will quite often be well stained)
4.  Rinse the sections in water to remove any precipitate.
5.  Dip the section for one to two dips in an eosin counter stain ( I use 0.5% eosin in 70% alcohol      with a drop or two of acetic acid)
6.  Rinse in two changes of 100% alcohol, clear in two changes of Xylene and mount

Ganglia stain vivid Blue against a red or pink background.

Substrate  solution
Vernol buffer pH 7.4                            --- 2.0 ml
Nitro Tetrazolium Blue (sigma N6876) --- 2.0 mg
Distilled water                                     --- 2.0 ml
Add above solution to a 5.0 mg vial of T.P.N.H (sigma N1630) and use immediately

Vernol buffer pH 7.4
Sodium Barbitol  -- 1.0 gm in 100mls distilled water
Con HCl  --  4.0 mls in 1000 mls distilled water
Mix 56ml of the sodium barbitol solution with 44 mls of the HCL solution, and it is usually right on pH 7.4

Down in the States you may have problems getting the sodium barbitol, so if you do, I would think that most any other similar buffer at pH 7.4 would work as well.

Kerry Beebe A.R.T.
Histology Supervisor
Kelowna General Hospital
Kelowna B.C. Canada

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