Histotechnology Technical methods

Enzyme Histochemical Stain

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NADH DIAPHORASE

(MTT METHOD)

SECTIONS

Unfixed Cryostat Muscle Biopsy


SOLUTIONS

Solution a. Dehydrogenase stock (Can be made in bulk x20)

--MTT (1mg/ml) 2.5ml (50ml)

--0.2M Tris buffer pH 7.4 2.5ml (50ml)

--0.05M Magnesium chloride 1.0ml (20ml)

--Distilled water 2.5ml (50ml)

--0.5M Cobalt Chloride 0.5ml (10ml) --Adjust to pH 7.0 - store at -20 degrees centigrade

Solution b. Incubating solution --Soln.a. (Dehydrogenase stock) 0.9ml

--Distilled water 0.1ml

--co-enzyme NADH 2mg


METHOD

1. Incubate in solution b. for 90 minutes at 37ÉC.

2. Fix in 4% formalin for 15 minutes

3. Wash well in distilled water.

4. Mount in phosphate buffered glycerine jelly.


RESULTS

NADH diaphorase activity - blue/black


Notes

NADH DIAPHORASE (NADH TETRAZOLIUM REDUCTASE)

The 'diaphorases' are dehydrogenase enzymes which catalyze the dehydrogenation of the reduced forms of the co-enzyme NAD and NADP, i.e. they catalyze the reactions.

NADHææææææææææÆNAD++H NADPHæææææææææÆNADP++H

The hydrogen liberated then combines with the tetrazolium salt, e.g, Nitro Blue Tetrazolium - (N.B.T.) or M.T.T. to form a formazan salt which is visible at the site of enzyme reaction.

NADH NADH æææææÆ NAD + H Tetrazolium diaphorase [omega]__________[omega] [omega] Formazan

The MTT in the dehydrogenase stock may be replaced by NBT.

--NBT (4mg/ml) 2.5ml

--0.2M Tris or phosphate buffer (pH 7.4) 2.5ml

--0.05M Magnesium chloride 1.0ml

--Distilled water 3.0ml

--Adjust to pH 7.0

This method enables the sections to be dehydrated, cleared and mounted. A rinse in acetone after fixation of the reaction method will remove soluble pink formazan deposit.

NOTE: No responsibility is assumed by The University of Nottingham or the Queens Medical Centre NHS Trust for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. It is the users responsibilty to ensure that all procedures are carried out according to appropriate Health and Safety requirements.


© Copyright 1997 University of Nottingham Medical School Division of Histopathology. This page was last built on Mon, May 5, 1997 with Frontier. Thanks for looking in. Comments to James.Lowe@nottingham.ac.uk