AW: [Histonet] CAE and Mature Mast cells look at procedure

[Gudrun Lang]

 We use the same procedure, but instead of phosphate buffer we take
veronalbuffer.
In summer, in our old lab, we often had problems with this stain. We thought
it was because of the "summer-heat". Now in the klimated new lab, it works
constantly.

I always shake the fuchsin-natriumnitrit-solution rather rigid, until the
"chloro"-smell disappears.
After adding the buffer, the solution must be yellowish. With a red tinge,
it doesn't work.
Gudrun

-----Ursprüngliche Nachricht-----
Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Madary,
Joseph
Gesendet: Montag, 24. Juli 2006 18:22
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [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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