Fluorescent staining for Callose with aniline blue v1

Fluorescent staining for Callose of sieve tubes of phloem. Calose (β-1→3-glucan) is the distinguished polysaccharide present on the sieve plates, of each sieve tube member of phloem tissue. Aniline blue stain specifically callose and it is use as a marker for identification of sieve elements of phloem. PROCEDURE – STAINING FOR CALLOSE WITH ANILINE BLUE (modification of Klaus Herburger and Andreas Holzinger (2016). Prepare Sørensen’s phosphate buffer (0.1 M, pH=8.0) according with Ruzin (1999). Stock solutions. A) 0.2 M of NaH2PO4 (Sodium phosphate monobasic anhydrous, CAS Number: 7558-80-7) BioUltra, ≥99.0% (T) (Sigma-Aldrich S3139). B) 0.2 M of Na2HPO4 (Sodium phosphate dibasic anhydrous, CAS Number: 7558-79-4) Ultra ≥99.5%, (Sigma-Aldrich S7907). To prepare 100 mL of the buffer (0.1 M, pH=8.0), mix 2.7 mL of stock solution (A) with 47.3 mL of stock solution (B) and adjust volume to 100 mL in a Schott flask. Add 0.5 % Aniline blue (w/v) to freshly prepared Sørensen’s phosphate buffer. Fix tissue in 2.5% glutaraldehyde in phosphate buffer Sorensen's solution (0.1 M, pH 7.2) and processed for LR white resin embedding according with manufactures. Obtain transverse semi thin sections (1 µm) with an ultramicrotome and bond the sections to microscope grass slides with heat (60 C) during 15 min. Stain sections with 0.5 % of Aniline blue (CAS Number: 28631-66-5, Sigma Aldrich, Germany) in Sørensen’s phosphate buffer (0.1 M, pH=8.0) for 1 h under low light intensities (~5 μmol photons m-2 s-1) to prevent degradation of the dye. Observe tissue under an Epifluorescence Microscope (Axioscope 2 Plus, Carl Zeiss AG) using a Set 01 (excitation: band pass (BP) 365/12 nm; emission: long pass (LP) 397 nm) and connected to an Axiocam MRc5 camera. Calose of sieve elements stain bright green yellow. References Herburger, K., & Holzinger, A. (2016). Aniline blue and Calcofluor white staining of callose and cellulose in the streptophyte green algae Zygnema and Klebsormidium. Bio-Protocol, 6(20), e1969. http://doi.org/10.21769/BioProtoc.1969