Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 35

3. Stain in the solution for minimum of 1.5 h at laboratory tem-
perature (the time can be extended signifi cantly without a risk
of overstaining; warming the staining solution up to 60 °C
might accelerate the staining process).
4. Quickly rinse excess of dye solution with detergent solution
(e.g., 0.5 % aq. solution of SDS).
5. Carefully wash with water.
6. Mount into 75 % aq. glycerol.
7. Sudan red gives intense red coloration of lipidic compounds,
while Fluorol yellow yields green/yellowish fl uorescence with
UV excitation (for possible pitfalls see Note 4 ).

3.10 Berberine:
Toluidine Blue Staining

1. Stain sections (we preferentially use fresh ones after aldehyde
fi xation) for at least 1 h in Berberine solution.
2. Wash twice with water.
3. Counter stain in 0.05 % toluidine blue O in water for 5-10 min.
Alternatively Crystal violet (syn. Gentian violet) can be used to
effi ciently quench background fl uorescence.
4. Wash carefully with water.
5. Mount into water or 25-50 % glycerol.
6. Observe under UV excitation as a yellow fl uorescence or
under blue excitation as a green emission.

3.11 Wiesner's
(HCl: Phloroglucinol)
Reaction

1. Stain the sections (we preferentially use fresh ones, but paraf-
fi n-embedded sections might be used after rehydration) at
laboratory temperature with acid phloroglucinol solution till
the sections turn red (within few minutes).
2. Mount the sections into glycerol acidifi ed with sulphuric acid
to maintain the reaction product, which last for several days or
even weeks. Hydrochloric acid (escaping hydrogen chloride)
is highly aggressive to metallic and optical parts of the micro-
scope. That is why it is strongly recommended not to use it in
close vicinity of the microscope.
3. Observe with bright fi eld optics. Lignin modifi ed cell walls
stain cherry red.

1. Treat the section with aniline sulphate solution for 5 min in
lab temperature.
2. Mount into acidifi ed glycerol described for Wiesner's reaction.
3. Observe in bright fi eld. Lignins are stained bright yellow.

3.12 Aniline
Sulphate Procedure

3.13 Mäule Reaction
for Lignin

1. Oxidize sections in solution of KMnO 4 for 10-20 min.
2. Wash 3× with distilled water.