Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 31

Centrifuge to remove undissolved propyl gallate. Solution can
be stored in dark at 4 °C for about a year.
7. TRIS buffer (0.1 M, pH 9.0): Dissolve 12.1 g TRIS base in
approx. 750 ml of distilled water. Titre with 1 M HCl to pH
9.0, and fi ll with distilled water to 1 l of fi nal volume.
8. High-humidity chamber is used to prevent evaporation of low
volumes of antibodies from slides. Simple chamber can be
made of large Petri dish with soaked tissue or fi lter paper on
the bottom. Glass rods are used to separate slides from the
soaked tissue and prevent their contact.

1. Berberine dye solution: 0.2 % Berberine hemisulphate in water.
The solution is close to the saturation and crystals will form
when stored at 4 °C, those should be redissolved before use.
0.1 % solution is used in most publications but higher concen-
tration does not cause overstaining.
2. Toluidine blue O dye solution: 0.05 % w/v of toluidine blue
O in water.
3. Crystal violet solution: 0.05 % w/v Crystal violet in water.

2.4 Berberine:
Toluidine Blue Staining

2.5 HCl:
Phloroglucinol
(Wiesner's Reagent)

1. Acidifi ed phloroglucinol solution: Phloroglucinol (1 % w/v,
saturated) solution in 18 % aq. HCl. The solution oxidizes
with time, turns deep yellow-brown and the intensity of reac-
tion decreases. That is time to change it for fresh one.
2. Acidic glycerol to mount sections: Mix 75 % (fi nal volume) of
glycerol with 15 % of H 2 O and 10 % of concentrated H 2 SO 4 .

2.6 Mäule Reaction

1. KMnO 4 solution: Prepare fresh 1 % w/v aqueous solution of
KMnO 4 .
2. Alkalized glycerol: 15 % (w/v) solution of Na 2 CO 3 in 50 %
aqueous glycerol (alternatively 15 % ammonium hydroxide in
75 % aq. glycerol can be used).

Two optional co-substrate mixtures are described. 2.7 Peroxidase

Activity Detection

1. DAB reaction mixture: Prepare fresh solution just before incu-
bation, containing 500 μ l DAB stock (1 mg in 1 ml of distilled

water, see Note 1 ), 499 μ l acetate buffer (pH 5; 0.1 M), 50 μ l

NiCl 2 (8 % w/v in distilled water). Add 1 μ l of H 2 O 2 (30 % in

distilled water) just before usage.
2. TMB reaction mixture: Prepare fresh solution just before
incubation, containing 10 μ l TMB stock (10 mg in 1 ml 96 %

ethanol) and 989 μ l acetate buffer (pH 5; 0.1 M). Add 1 μ l of

H 2 O 2 (30 % in distilled water) just before usage.