Aniline Blue and Calcofluor White Staining of Callose and Cellulose in the Streptophyte Green Algae Zygnema and Klebsormidium

Materials and Reagents

1. 2 ml Eppendorf tubes
   
2. Epoxy coated microscopic slides with 8-12 wells (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 1014326410EPOXY ) and cover slips
   
3. Algal cells or filaments (Figure 1A)
   Note: In principle, this protocol is not restricted to green algae and can be applied to all plant cells surrounded by a cell wall, including cell cultures.
   
4. Sodium hydroxide pellets (NaOH) (EMD Millipore, catalog number: 106469 )
   
5. A. bidest. (double distilled water)
   
6. Sodium phosphate monobasic dihydrate (NaH₂PO₄·2H₂O) (EMD Millipore, catalog number: 1063420250 )
   
7. Sodium phosphate dibasic dihydrate (Na₂HPO₄·2H₂O) (EMD Millipore, catalog number: 1065800500 )
   
8. Culture medium (BBM [Bischoff and Bold, 1963], MBBM [Starr and Zeikus, 1993])
   
9. Aniline blue diammonium salt (Sigma-Aldrich, catalog number: 415049 )
   
10. Calcofluor white (Sigma-Aldrich, catalog number: 18909 )
    Note: Adding Evans blue as a counterstain to diminish background fluorescence is not obligatory, since background fluorescence in the algae investigated is very low.
    
11. 0.2 M NaH₂PO₄·2H₂O stock solution (see Recipes)
    
12. 0.2 M Na₂HPO₄·2H₂O stock solution (see Recipes)
    
13. 50 ml of Sørensen’s phosphate buffer (see Recipes)
    

Equipment

1. Glass Pasteur pipettes
   
2. 25 ml beaker
   
3. 1,000 ml and 500 ml volumetric flask
   
4. 100 ml Schott flask
   
5. Balance
   
6. Pair of fine-pointed tweezers
   
7. Metal rack (1.5 ml microfuge rack) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 3166185 )
   
8. Water bath or heat plate and beaker
   
9. Tabletop centrifuge (Sigma Laborzentrifugen, model: Sigma 1-14 )
   
10. Shaker (Thermo Fisher Scientific, Thermo Scientific^TM, model: Compact Digital Microplate Shaker)
    
11. Epifluorescence microscope capable of UV excitation and long pass detection, with high numerical aperture lens, connected to a camera for documentation (e.g., We used a ZEISS Axiovert 200M equipped with a 63 x 1.4 NA objective, a OSRAM HBO 50 Q/AC L1 CZ Mercury short ARC Photo optic lamp, Zeiss Filter Set 01 (excitation: band pass [BP] 365/12 nm; emission: long pass [LP] 397 nm and connected to a Axiocam MRc5 camera) (Carl Zeiss, model: Axiovert 200M )
    

Procedure