This procedure is intended for detecting polysaccharides in tissues. It is also a good staining for basal membranes and cartilage tissue. Any fixative is suitable for this staining.
The staining by Schiff reagent solution requires a previous step where chemical modification of the tissular polysaccharides takes place, that is, histochemistry. The periodic acid performs this histochemical modification.
Procedure
Samples are fixed, embedded in paraffin, and 8 µm thick sections are obtained. Sections are mounted onto gelatin coated slides.
1.- 2x10 min 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.- 0.5 % peryodic acid in distilled H2O, 10 min
The C-C bonds of polysaccharides are oxidized in this step, and aldehyde groups are formed.
8.- Distilled H2O, 3x1m
9.- 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.
10.- Tap H2O, 5 min
11.- Distilled H2O, rinses
12.- Mayer's hematoxylin, 5 min
13.- Tap H2O, 15 min
14.- Distilled H2O, 5s
15.- 80º ethanol, 5 min
16.- 96º ethanol, 5 min
17.- 100º ethanol, 2x10 min
18.- Xylene, 2x10 min
19.- Mounting medium and coverslipped
Results
Polysaccharides: strong red to fuchsia
nuclei: dark 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
Xyleno
50º, 70º, 80º, 90º, 96º y 100º ethanol
Periodic acid, 5 % in distilled H2O
Schiff reagent solution
Mayer's hematoxylin
Distilled H2O
Tap H2O
Mounting medium
Labware
Staining dishes
Slides racks
Coverslips
