Scott's CEC Technique
for Acid Mucopolysaccharides

Scott and Dorling's critical electrolyte concentration method for acid mucopolysaccharides.

Solutions

Alcian blue pH 5.8
Alcian blue	0.05	g
Acetate buffer pH 5.8	100	mL
Magnesium chloride	as	specified below

Preparation
To the alcian blue solution add the following amounts of magnesium chloride to obtain the electrolyte molarity noted. Prepare fresh.

0.06	M	1.220	g
0.2	M	4.066	g
0.3	M	6.099	g

0.4	M	8.132	g
0.5	M	10.165	g
0.6	M	12.198	g

0.7	M	14.231	g
0.8	M	16.264	g
0.9	M	18.297	g

Neutral red, 0.5% aqueous.

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable.

Method

1. Bring sections to water via xylene and ethanol.
2. Place a section into each alcian blue electrolyte concentration chosen and leave overnight.
3. Wash with water.
4. Counterstain with neutral red, if wished.
5. Dehydrate with ethanols.
6. Clear with xylene and mount with a resinous medium.

Expected results

Electrolyte concentration
0.06	M	–	All acid mucopolysaccharides blue
0.2 - 0.3	M	–	Sulphated acid mucopolysaccharides blue
0.5 - 0.6	M	–	Strongly sulphated acid mucopolysaccharides blue
0.7 - 0.8	M	–	Heparin, heparan sulphate, keratan sulphate blue
0.9	M	–	Keratan sulphate blue
Nuclei		–	red, if counterstained

Notes

1. The amounts given for magnesium chloride are based on a formula weight of 203.3 for the hexahydrate. If the salt used is other other than this, adjust the amounts added to each solution to obtain the correct molarity.
2. Magnesium chloride is hygroscopic. Store appropriately.
3. The electrolyte molarities for each mucopolysaccharide are not considered absolutes, and personal standardising and evaluation are required.
4. This method is usually explained on the basis of magnesium ions competing with alcian blue for binding sites on mucopolysaccharides. As the concentration of magnesium ions increases, so more binding sites are blocked from access to alcian blue.

 

Reference
Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.