This procedure is intended for studying polysaccharides distribution in tissues, such as glycogen and mucopolysaccharides. It is also a good staining for basal membranes and cartilage. Any fixative can be used with this staining procedure.
PAS and Alcian blue are combined to distinguish acid mucopolysaccharides, stained in blue with Alcian blue, from other polisaccharides, stained in pink-redish with the PAS reactive.
Procedure
Samples are fixed, embedded in paraffin, and 8 µm thick sections are obtained. Sections are mounted onto gelatin coated slides.
1.- 2x10 min in xylene
2.- 100º ethanol, 2x10 min.
3.- 96º ethanol, 10 min.
4.- 80º ethanol, 10 min.
5.- 50º ethanol, 10 min.
6.- Distilled H2O, 5 min.
7.- 1 % Alcian blue, 30 min.
Alcian blue:
1 g Alcian blue (C.I. 74240)
3 ml acetic acid
97 ml distilled water
8.- Tap H2O, 3 min.
9.- 0.5 % peryodic acid in distilled H2O, 10 min.
10.- Distilled H2O, 3x1m.
11.- Schiff reagent solution for 30 min, in dark. The time may change depending on the temperature and how many times the Schiff reagent has been used. It can be even 10 min.
The aldehyde groups react with the Schiff reagent solution (which contains sulfurous acid and fuchsine) and gives a red-fuchsia coloring. Section background is pale pink.
A step with 0.5 % of potassium (sodium) metabisulphite for 2 min can be done to remove the traces and precipitates of Schiff reagent remaining in the tissue.
12.- Tap H2O, 5 min.
13.- Distilled H2O, rinses.
14.- Mayer's hematoxylin, 5 min (optional).
15.- Tap H2O, 5 min (if hematoxylin, optional).
16.- Distilled H2O, 20s (optional).
17.- 80º, Ethanol 5 min.
18.- 96º ethanol, 5 min.
19.- 100º ethanol, 2x10 min.
20.- Xylene, 2x10 min.
21.- Mountin media and coverslipped.
Results
Polysaccharides: strong pink-fuchsia
Acids mucopolysaccharides: blue
Notes
How long is the Schiff reagent solution step depends on the temperature and how fresh is the solution. Microwaves can be used to decrease the time of incubation.
The schiff reagent solution can be used many times, but it must be stored in the refrigerator. It is warmed up to room temperature before each staining procedure.
The schiff reagent solution must keep a clear-yellow color, and it should be discarded when the color turns pink. If white precipitate is observed, it can be dissolved again by stirring.
Products
Xylene
50º, 80º, 90º, 96º, 100º ethanol
Alcian blue (C.I. 74240)
Peryodic acie
Schiff reagent solution
Mayer's hematoxylin (optional)
Acetic acid
Distilled H2O
Tap H2O
Mounting medium
Labware
Staining dishes
Staining racks
Coverslips
