Re: Leder Stain

From:=?iso-8859-1?q?Steve=20Machin=20UK?=

Her is a copy of our labs method for Leder's

Best Wishes
Steve Machin UK

Introduction	

Frozen or 4 ėm paraffin sections to demonstrate Mast cells and cells
of granulocytic lineage.

Method

1.	Fix cryostat section in buffered formalin or take paraffin
sections to water*.
2. In a 50 ml beaker take 4 drops 4% Pararosaniline hydrochloride
solution, add 4 drops freshly prepared 4% Na nitrite solution, mix 1
min, the mixture should turn straw coloured.
3. Add 20 ml Veronal acetate buffer solution pH 9.1 adjust to pH 6.3
using N HCl
4. Weigh out 0.01g Naphthol AS-D chloroacetate and dissolve in 0.5 ml
Dimethyl formamide. 
5. Add this solution to the Pararosaniline/Buffer mixture, the
resulting solution
	should turn flocculent pale pink in colour.
6.	Filter onto slides and leave 20-30 min at R.T.
7.	Wash in water.
8.	Lightly counterstain in Haematoxylin.
9.	Wash in water, do not differentiate, blue in Scott’s tap water.
10.	Dehydrate clear and mount in picolyte (COSHH H I) F.

*Dewax in Xylene (COSHH H I) F, rehydrate by washing in 99% alcohol
(COSHH H I) HF
  then 95% alcohol then water.


Results	
	
	Chloroacetate esterase activity	-  red
	(Present in cells of myeloid series, granulocytes onwards).
	
	Nuclei 		-blue


Control :-  MAST CELLS


Limitations of Method

Early cells of the myeloid series may not react.

 Reagents/ Solutions

Veronal Acetate Stock Solution.

	Na acetate (trihydrate) 1.94 g or 1.2 g (anhydrous) (COSHH I)
	Na di-ethyl barbiturate (sodium barbitone) (COSHH H I) 2.9 g
	Distilled water 100 ml

Veronal Buffer pH 9.1

	Veronal acetate stock soln.    50.0 ml
	Distilled water                       197.5 ml
	0.1N HCl (COSHH C H I)        2.5 ml

Pararosaniline Solution

	Equal amounts of a and b (i.e. 4 drops of each using a 1 ml
disposable pipette).
	(a) 4% Pararosaniline (Basic Fuchsin) (COSHH Tc)  in 20% HCl (COSHH
C H I)
	(b) 4% Na nitrite (0.04 g/ml)  (COSHH T I)


	To prepare 4% Pararosaniline stock (a)
2 g of Pararosaniline + 40 ml of distilled water, heat until almost
boiling, cool to room temperature and add 10        ml of  HCl.
Filter into bottle.
 COSHH

Hazardous substance
Xylene Harmful Irritant
Wear nitrile gloves and only handle in special extraction cabinet,
wear goggles when decanting.

99% and 95% alcohol (Industrial meths) Harmful Irritant
Wear gloves when handling.

Basic Fuchsin (Pararosaniline) (as dry powder) Toxic Carcinogen
Harmful
N.B. Basic Fuchsin is particularly "dusty", wear gloves when handling
solutions, wear face mask when weighing out dye and make up solutions
in fume cupboard.

Sodium barbitone Harmful Irritant
Wear gloves when handling.

Hydrochloric acid (conc.) Corrosive Harmful Irritant
Wear gloves and handle in fume cupboard.

Sodium nitrite Toxic Irritant
Wear gloves when handling. 

Picolyte mountant (contains Xylene) Harmful Irritant
Wear nitrile gloves, and only handle in special extraction cabinet.


N.B. After any powder is weighed out, it must be put into the solute
straight away. It must not be transferred from the weighing room to
another room as a powder.
After the stain/ chemical has been made, the balance and surrounding
work bench must be wiped clean.




FIRE HAZARD

Hazardous substance 
Xylene Flammable
Store in Flammable Bin and replace container immediately after use.

99% and 95% alcohol (Industrial meths). Highly Flammable
Store in Flammable Bin and replace immediately after use.

Picolyte mountant (contains Xylene) Flammable
Store stock bottle in Flammable Bin and return container immediately
after use.                                        



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