Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 29

2 Materials

All solutions should be prepared in distilled water unless stated
otherwise.

2.1 Various Single-
Component Dyes

1. Toluidine blue O: 0.01-0.025 % (w/v) toluidine blue O in
water. Store at 4 °C. It can last for long rather time (months).
Check periodically for mold.
2. Calcofl uor white stock solution: 1 % solution of Calcofl uor in
distilled water. Gently heat the solution and add minimum of
1 M sodium hydroxide (fi nal pH 10-11) to dissolve the dye
completely. Aliquots of stock solution can be stored at −20 °C
for a long period of time.
3. Alcian Blue: 0.1 % (w/v) Alcian Blue in 3 % acetic acid (alter-
natively citrate buffer of pH 3.5, 100 mM can be used, but is
less selective).
4. Ruthenium red: 0.05 % (w/v) aqueous solution. Do not use
phosphate and some other anionic buffers as those might pre-
cipitate the dye.
5. Aniline blue fl uorochrome: 0.005-0.01 % solution of water-
soluble aniline blue buffered to pH above 8.5 (e.g., 100 mM
K 2 HPO 4 with pH 9). Stock solution of purifi ed aniline blue
fl uorochrome Sirofl uor (1 mg/ml) in distilled water can be
stored in aliquots at −20 °C.
6. Resorcin blue: Dissolve 3 g of resorcinol (p.a.) in 200 ml of
distilled water. Add 3 ml of concentrated ammonia. Heat up
in steam bath for 10 min (do not boil!). Let the red-brown
solution cool down to lab temperature. The solution will
gradually gain blue color (after approx 6 h). Heat again in the
steam bath for about 30 min till no more ammonia escapes
(test with wet pH indicator paper). Dilute prepared solution
1:50 with distilled water for staining.
7. Sudan red 7B and Fluorol yellow: Dissolve Sudan red 7B
(0.1-0.2 % w/v) or Fluorol yellow 088 (0.01 % w/v) in PEG-
400 heating the solution up to 90 °C. Do not allow to go over
100 °C as overheating change staining properties. Add equal
volume of 90 % aq. glycerol. Filter solution through coarse
fi lter paper or let stand overnight, and decant supernatant or
centrifuge to sediment crystals of undissolved dye (if present it
contaminates surface of sections).
8. Aniline sulphate solution: Dissolve 1 g of aniline sulphate
(toxic and dangerous to environment) in 10 ml of 0.05 M
H 2 SO 4 and 90 ml of 70 % EtOH.