[Histonet] CAE and Mature Mast cells look at procedure

[Tony Henwood]

Several comments:

Leave the New Fuchsin and 4% Sodium Nitrite solution for 2-3 minutes before adding to the buffer. (The solution should lighten after some bubbling).

Stain the sections longer than the 20 minutes. Check microscopically.

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 <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Madary, Joseph
Sent: Tuesday, 25 July 2006 2:22 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] CAE and Mature Mast cells look at procedure


Okay my histoheroes and heroines,
One of the investigators here asked for a CAE designed for plastics(but supposedly works on FFPE).  I did this protocol, but it isn't working. I am having a rough time because I am not used to making mistakes(kidding).  She needs mature mast cells only, I gave her 7 different Mast cell procedures that all worked very well(even the dominicis), and one Alcian Blue Safranin that stains Heparin containing Mast cells red Amine containing Mast cells blue. So 2 questions are:

1-Do mature cells stain differently?
2-Does anyone have a decent CAE they can share?  Here is the one I used and the control is fresh rat skin and all the other procedures worked on that block. 

	Chloroacetate Esterase (CAE) Staining

Preparation of Solutions

1.	New Fuchsin Solution
			New Fuchsin				1g
			2N hydrochloric acid (HCl)	          25ml

2.	4% Sodium Nitrite Solution
			Sodium Nitrite				1g
			ddH2O                                               25ml

3.	Phosphate Buffer (0.1M, pH 7.6)
		Stock Solutions
A.	0.2M solution of monobasic soduim phosphate (27.8g NaH2PO4 in 1L H2O)
B.	0.2M solution of dibasic sodium phosphate (28.4g Na2HPO4 in 1L H2O)
      --> To make pH 7.6 Solution: mix 6.5mL of A, 43.5mL of B, and 50mL of ddH2O.

4.	Naphthol AS-D Chloroacetate Solution (Store at -20°C)
		Naphthol AS-D Chloroacetate		10mg		100mg
		N-N Dimethyl Formamide		              5ml		  50ml


Staining Solution (make fresh before each use)

In a single vial:	1) mix New Fuchsin and 4% Soduim Nitrite
			2) add phosphate buffer and mix
			3) add Naphth AS-D Chloroacetate Soultion and mix well

	Use 1 ml freshly mixed solution for approx 4 tissue slides
New Fuchsin				2.5µl		5µl		12.5µl		25µl
4% Sodium Nitrite			2.5µl		5µl		12.5µl              25µl
Phosphate Buffer	                           1ml               2ml                      5ml             10ml
Naphthol AS-D Chloroacetate	  50µl	         100µl		  250µl	           500µl

Staining Procedure
1.	Stain with fresh staining solution for 20 min. 
2.	Wash with running tap water (For plastic sections use wet cotton swab wipe off dusty residue).
3.	Counterstain with Gill's II hematoxylin for 20 to 45 seconds (or methyl green for 30 seconds).
4.	Wash with warm tap water to blue  (longer will result darker blue, or dip into 50% of lithium carbonate for 5 - 20 seconds for deeper blue, then rinse with water).
Dry and mount with mounting medium. (For paraffin sections, quick dehydration before mounting).

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