3.10　 Visualization of cell wall with calcofluor

Tomomichi Fujita

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

Solution preparation

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

Stock solution　

fluorescent brightener 28 (sigma、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 a stock solution into one hundredth (10 ug/ml)

　　 1 mg/ml fluorescent brightener 28　　　　　　　　　 10 ul　　

　　 H2O　　　　　　 　　　　　　　　　　　　　　　　　 1 ml　　

　　 Store at –20˚C.

Protocol

Drop the calcofluor working solution onto tissues or cells, and observe under UV excitation. Excess amount of calcofluor, which causes high background should be washed with PBS buffer. Wash cells as quick as possible, otherwise cells are subject to plasmolysis.

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