Alexa fluor 488 antibody

Immunofluorescence-based detection of H3 histones phosphorylated at the conserved serine 10 residue was performed according to the methods described in [23 (link)]. Apical parts of V. faba roots (1.5-mm-long) excised from the untreated (control) and all cadmium-treated seedlings were fixed for 45 min (20 °C) in PBS-buffered 3.7% paraformaldehyde, washed with PBS, and placed for 45 min (37 °C) in a citric acid-buffered digestion solution (pH 5.0) containing 2.5% cellulase (Onozuka R-10; Serva, Heidelberg, Germany), 2.5% pectinase (Fluka, Germany), and 2.5% pectolyase (ICN, Costa Mesa, CA, USA). After washing with PBS and distilled water, root meristems were squashed onto slides and air dried. Slides were pretreated for 50 min with PBS-buffered 8% bovine serum albumin (BSA) and 0.5% Triton X-100 (20 °C) and incubated for 12 h in a humidified atmosphere (4 °C) with rabbit polyclonal anti-H3S10Ph antibody (1:500; Sigma-Aldrich, Poznan, Poland), dissolved in PBS containing 1% BSA, according to methods described earlier [23 (link),25 (link)]. After washing with PBS, slides were incubated for 1.5 h (20 °C) with secondary goat anti-rabbit conjugated to Alexa Fluor^® 488 antibody (1:500; Cell Signaling) dissolved in PBS (1:500) and counterstained with propidium iodide (PI; 0.3 mg mL⁻¹). Slides washed with PBS were air dried and embedded in a PBS/glycerol/DABCO mixture.