Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 34

3. Mount into water or 50 % glycerol (alternatively it is possible
to observe directly in staining solution due to low fl uorescence
of unbound fl uorochrome in water solutions).
4. Optionally the background autofl uorescence can be decreased
counterstaining Sirofl uor with toluidine blue or Crystal violet
(as described for Berberine staining).
5. Observe in fl uorescence setup. Fluorochrome yields yellow-
green fl uorescence with blue excitation and pale yellow
fl uorescence with UV excitation.

3.7 Resorcin Blue
Staining

1. Dilute prepared solution 1:50 with distilled water and stain
sections for 1-2 min.
2. Carefully wash 3× in water.
3. Mount into citrate buffer pH 3.2 (or 50 % buffered glycerol).
4. Observe in bright fi eld optics. Callose is stained blue while
lignifi ed structures change the color to red.

3.8 Callose
Immunodetection

1. Fix pieces of tissue in −20 °C methanol for 5-10 min.
2. Wash 2 × 5 min v PBS.
3. Prepare sections, preferentially fresh hand sections. Cryo-
sections dried to slides should be rehydrated for 30 min in
PBS. If paraffi n sections are used, paraffi n should be removed
from sections in toluene, rehydrated via alcohol series down to
water, and PBS.
4. Block the nonspecifi c protein binding with 1 % casein solution
in PBS, 15 min.
5. Wash in PBS for 2 min and blot excess of solution from the
edge of slide.
6. Apply primary antibody in high-humidity chamber at labora-
tory temperature for 2 h (time of application might be
prolonged).
7. Wash 2 × 10 min in PBS, blot excess of solution.
8. Apply secondary antibody in humidity chamber at laboratory
temperature for 2 h.
9. Wash 2 × 5 min in PBS.
10. Counter stain with toluidine blue for 10 min.
11. Mount into glycerol with propyl gallate.

1. Fresh hand sections or cryotome sections are best suitable for
the staining.
2. Blot sections to minimize transfer of water into staining solu-
tion, optionally 75 % glycerol wash might be included before
staining to minimize precipitation of dye.

3.9 Sudan Red 7B
or Fluorol Yellow
Staining Procedure