3.6　 Visualization of cell walls with calcofluor

Tomomichi Fujita and Takashi Murata

Calcofluor binds cellulose in cell walls and is used to visualize cell walls. It emits blue-white fluorescence under UV excitation.

Solution preparation

1. 1 mg/ml fluorescent brightener 28 (calcofluor)

Stock solution　　　　

fluorescent brightener 28 (sigma, F-3397)　　　　　　　 　　　　　　　 1 mg　　　

H₂O　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　 1 ml　　　　

　 Dissolve, then store at -20˚C covered with aluminum foil to prevent light.

2. fluorescent brightener 28 solution

　 Dilute the stock solution one hundred-fold (10 ug/ml)

　　 1 mg/ml fluorescent brightener 28　　　　　　　　　　 10 ul　　　

　　 H₂O　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　 1 ml　　　　

　　 Store at –20˚C.

Protocol

Place tissues or cells onto a glass slide, and drop the calcofluor working solution onto them. Wash the preparation with PBS. Replace PBS with distilled water as quickly as possible and observe under UV excitation immediately.

(Figure) The first asymmetric cell division of a P. patens protoplast. Cells in bright field (upper) and with calcofluor under UV field (lower).