Histotechnology Technical methods

Enzyme Histochemical Stain

Methods Index

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SUCCINATE DEHYDROGENASE

SUCCINATE DEHYDROGENASE (MODIFIED FROM PETTE & TYLER, 1983)

1. Phosphate buffer (100mM, pH 7.6)

--Solution A (1.36g KH2PO4 /100ml) 12ml

--Solution B (1.42g Na2HPO4/100ml) 88ml

2. NBT Stock

--Phosphate buffer as above 100ml

--KCN 6.5mg

--EDTA 185mg

--Nitroblue tetrazolium (NBT) 100mg

Freeze in 2ml aliquots

3. Succinate stock (500 mM sodium succinate)

--Sodium succinate 2.7g

--Distilled water 20ml

Freeze in 5ml amounts

4. Incubation medium

--NBT stock 2.0ml

--Succinate stock 0.2ml

--Phenazine methosulphate 0.7mg (one small crystal)

Mix just before use, keep out of strong light.


SECTIONS

Fresh cryostat on coverslips


INCUBATION

Rat tibialis anterior, soleus Room temp/5 minutes

Human quadriceps, gastrocnemius 37ÉC/10-15 minutes

Wash in distilled water, fix for 15 minutes in formol-calcium. Dehydrate, clear and mount. (Alternatively, a brief rinse in acetone will remove pink monoformazans, then mount in Apathy's medium or glycerin-jelly).


RESULT

sites of SDH activity (mitochondrial) stain blue.

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