Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 32

3. Acetate buffer (pH 5; 0.1 M): Mix 14.8 ml of 0.2 M acetic
acid with 35.2 ml 0.2 M sodium acetate and make up to
200 ml with distilled water.
4. Solutions for peroxidase inhibition: Use either (1) fresh solu-
tion of 3 % H 2 O 2 in methanol, (2) acetate buffer containing
0.1 % sodium azide and 0.5 % H 2 O 2 , or (3) acetate buffer con-
taining 0.1 % phenylhydrazine.

3 Methods

1. Stain fresh sections in toluidine blue O solution for 1-5 min.
2. Wash carefully in water.
3. Mount into water or low percentage glycerol (less than 25 %
aqueous solution) to maintain metachromatic staining
( see Note 2 ). If resin sections are used, let them air dry and
mount them with nonaqueous media.
4. Observe in bright fi eld optics.

3.1 Toluidine Blue
Staining

3.2 PARS Reaction
for Detection of Cell
Wall Polysaccharides

We most commonly use fresh sections. Other types of sections
should be fully hydrated before treatment.

1. Select parallel control section and skip H 5 IO 6 oxidation step
for those.
2. Oxidize sections in 1 % w/v H 5 IO 6 for 1 min in laboratory
temperature. Time should be adjusted properly if bigger
objects (wholemounts) are treated.
3. Wash sections in distilled water 3×.
4. Optionally apply for 3 min the reducing solution and wash
again with distilled water. Solution can be applied to clear
away remainings of periodic acid, not necessary for sections
but can be useful for bigger objects as wholemounts.
5. Stain in Schiff 's reagent for 10 min.
6. Wash very carefully in SO 2 water 3 × 10 min to prevent oxida-
tion of reduced colorless fuchsin and unspecifi c background
staining.
7. Mount into 50 % v/v glycerol in SO 2 water.
8. Purple coloration of the tissue is tightly bound, so it is also
possible to dehydrate objects and use permanent mounting.
The background staining strongly depends upon effi cient
Schiff reagent wash out.
9. Observe in bright fi eld. Presence of polysaccharides should be
indicated with purple coloration, compare with control sec-
tions. If indigenous aldehydes are present before periodic acid
treatment (in control sections), their reduction might be per-
formed in the beginning of procedure ( see Note 3 ).