4.5. F-Actin Ring Staining

To check the formation of F-actin rings (osteoclasts phenotypic characteristic), phalloidin was staining with fluorophore Alexa Fluor 488 (Life Technologies, Carlsbad, CA, USA), a mycotoxin from the group of phallotoxins produced by Amanita phalloides mushrooms. The structure has an affinity for actin filaments (COOPER, 1987). The cells were fixed with a 3.7% paraformaldehyde solution for 10 min and then washed with phosphate-saline buffer (English, Phosphate Buffered Saline—PBS) pH 7.4, and permeabilized with Triton X-100. Phalloidin staining was done at a ratio of 1:200 for 30 min. The fluorescence detection was excited/emitted at 495/518 nm, under a TSi Nikon fluorescence microscope.