Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 36

3. Flood with 1 M HCl until the dark precipitate disappears
(normally it takes about 30-60 s).
4. Wash gently with water.
5. Mount into alkalized glycerol.
6. Lignin is colorized red or brown red.

1. Fix the object in 4 % formaldehyde in phosphate buffer
(25 mM, pH 6.8) for 2-4 h at room temperature.
2. Carefully wash fi xative out of sections with phosphate buffer
(25 mM, pH 6.8) 2× for 15-20 min.
3. Prepare sections (we normally use hand sections) and select
parallel sections for controls.
4. Recommended controls are as follows: (1) sections treated
with reaction mixture without H 2 O 2 ; (2) sections with peroxi-
dase inhibited with H 2 O 2 in methanol, 10 min at laboratory
temperature; and (3) sections with peroxidase inhibited with
phenylhydrazine, 10 min at laboratory temperature.
5. Wash sections carefully with acetate buffer 2 × 5 min.
6. Treat the section with the incubation medium at 37 °C for 1 h
(or longer activity is weak).
7. Wash section carefully with acetate buffer 2 × 5 min.
8. Mount into 50 % glycerol.
9. Observe with bright fi eld optics.

3.14 Peroxidase
Activity Detection

4 Notes

1. DAB is commonly used as hydrochloride, which is more solu-
ble. If DAB is not in the form of hydrochloride it should be
dissolved fi rst in a drop of dimethylformamide and then add to
buffer. Low concentration of DMF (up to ~0.5 %) should not
affect peroxidase activity. DAB is carcinogenic. Waste should
be oxidized (commercial bleach, KMnO 4 ) before being dis-
carded. NiCl 2 catalyze precipitation of the product and decrease
its run from reaction site improving accuracy of localization.

2. Toluidine blue staining is very convenient for fresh sections.
Metachromasy is stable only in aqueous (highly polar) solu-
tions and disappears in organic solvents [ 44 ]. It fades even if
mount in stronger (we normally do not exceed 25 %) glycerol
solutions. The intensity of the staining (concentration of dye
solution) should be adjusted according to type and thickness
of the section. As thicker freehand sections might be over-
stained with presented dye concentration, it is reasonable to
dilute staining solution 5-10×. Other types of sections, e.g.,
paraffi n [ 45 ] or hydrophilic resin sections [ 46 ], work well if