RE: [Histonet] Enzyme staining Question

From:"Tony Henwood"



We use the fuchsin method and the fading is not apparent (ie fades as much as other techniques using fuschin). 
There is no need for an aqueous mountant, just dehydrate, clear and mount with a DPX type of mountant. Method as follows:

Non-Specific Esterase

Principle

Esterase is capable of hydrolysing carboxylic acid esters.  The technique is an azo-coupling method where the enzyme releases alpha-napthyl from alpha-napthol acetate, which then couples to para-rosanilin to produce and insoluble azo dye.

Fixation notes

1.	If tissue fixed in mercury containing fixative, do not remove any mercury pigment with iodine.
2.	For smears, fix for 2 minutes in absolute methanol containing 10% formaldehyde by volume and then wash.
3.	Do not overheat slides when drying.
4.	Do not fix tissue in an acid fixative, e.g. Zenker, Bowins.
5.	Do not decalcify tissue in acid solutions use EDTA.

Microtomy	
5ým paraffin sections
Solutions 
1.	Pararosaniline-HCl stock solution
Warning: Suspected Carcinogen - see MSDS

2.	4% sodium nitrite.
Warning: Toxic - see MSDS
Sodium Nitrite		0.8 g
Distilled water		20 ml

3.	Sorensen 0.1M Phosphate Buffer, pH 6.5
Sodium dihydrogen orthophosphate (MW 156)	2.648g
Disodium hydrogen orthophosphate (MW142)	1.135g
Distilled water						500ml

4.	Napthol AS-D Chloroacetate. (Sigma N-0758	1g)

5.	N,N - Dimethylformamide.
Warning: Suspected Carcinogen - see MSDS

6.	Harris Haematoxylin
 

Incubation solution

1.	Mix 0.2ml of pararosaniline solution (A), with 0.2ml of 4% sodium nitrate (B), mix and let stand for 1 minute.
2.	Add 35ml of phosphate buffer (C).
3.	Dissolve 0.01gm Napthol Chloroacetate (D), in 1 ml Dimethyl formanide (E).
4.	Mix solution prepared in step 2 with 3.


Controls

Both small intestine and kidney serve as good control material for non-specific esterases that are found in the lysosomes and endoplasmic reticulum of cells.

Method

1.	Deparaffinise and hydrate sections.
2.	Place slides into incubation solution and incubate for 30 minutes.
3.	Check microscopically if reaction is not complete, refilter and replace slides for 15-30 minutes more.
4.	Wash slides in water for 5 minutes.
5.	Counterstain in Harris's haematoxylin 2 min.
6.	Wash in tap water 3 min.
7.	Dehydrate, clear and mount.

Reference

1.	Leder, L.D., Klinische Wochenschrift, 1964, 42 (ii); 553.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318




-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Ford, Judi
Sent: Wednesday, 1 August 2007 7:53 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Enzyme staining Question


Hi Everyone,

 

First I want to thank you all for the information on the different stains for the myocardial ischemia. We have a plan to work with now and alternatives if the pathologist asks for more work.

 

My next question involves enzyme staining. I am doing a Naphthol AS-D Chloroacetate Esterase procedure that requires aqueous mounting media. This may be a dumb question but I was wondering if its better to do the procedure and examine right away or can the finished slide be examined at a later (day or two) without any fading artifact happening? 

 

Judi Ford

Roche Palo Alto

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