Mouse anti hdac3

HeLa cells were grown in a 6-chamber slide (LabTek) to 80% confluence, washed with 1× PBS, and fixed with 4% paraformaldehyde (Fisher) for 10 min. Cells were treated with TNBS buffer (0.1% Triton X-100, 1% FBS, and 0.1% NaN₃ in 1× PBS) for 20 min for permeabilization and blocking and then incubated overnight at 4 °C with one of the following primary antibodies: mouse anti-HDAC3 (Santa Cruz Biotechnology), rabbit anti-histone H1.3 (Abcam), or goat anti-Eg5 (Santa Cruz Biotechnology). Negative controls for all experiments were performed using non-immune IgG from the same species as the primary antibody. Following washes with 1× PBS, cells were incubated with secondary antibody: goat/donkey anti-mouse-FITC (Santa Cruz Biotechnology) or goat or donkey anti-rabbit-Texas Red (Santa Cruz Biotechnology) or donkey anti-goat-FITC or donkey anti-goat-TR for 1 h 15 min at room temperature. After the 1× PBS washes, chromosomes were stained with Hoechst (Invitrogen) for 5 min, and Prolong Antifade mounting medium (Invitrogen) was added before sealing the coverslip. The cells were imaged at ×1000 magnification using a Zeiss Axiovert 200 M optical microscope with confocal attachment, and the digital images were analyzed with ImageJ software.