Texas red anti mouse secondary antibody

For immunofluorescence on metaphase chromosomes, cell suspensions fixed in 3:1 methanol: acetic acid were dropped onto glass slides, allowed to dry incompletely and immediately immersed in PBS for 5 minutes at room temperature. Slides were washed in TEEN buffer (10 mM Triethanolamine- HCl pH 8.5, 2 mM EDTA, 250 mM NaCl) and blocked in 10% fetal calf serum (FCS) at 37°C for 10 minutes. Primary antibodies were added at the required dilutions and incubated in a humidified chamber at 37°C for 30 minutes. Slides were then washed in KB buffer (100 mM Tris-HCl pH 7.7, 1.5 M NaCl, 1% BSA). Secondary antibodies, raised in donkey and conjugated to fluorophores (Jackson Immuno Research), were diluted 1:500 in TEEN buffer, added to the slides and incubated at 37°C for 30 minutes. Slides were washed in KB buffer and stained in 50 μg / ml DAPI for 3 min at RT to detect DNA and nuclei. Slides were mounted in Vectashield (Vector Laboratories, Cat No H-1000) and imaged on a Zeiss Epifluorescence microscope using 100x objective. Primary antibody anti-H3S10p (1:100 dilution, clone CMA313) was a gift from Hiroshi Kimura (Hayashi-Takanaka et al., 2009 (link)), and detected with a Texas Red anti-mouse secondary antibody (1:500 dilution, Jackson Immuno Research). Anti-SMC2 antibody was detected with an FITC labebelled anti-rabbit secondary antibody (1:500 dilution, Jackson Immuno Research).

RMS cells were seeded overnight in 24-well plates. Cells were treated with 5-aza-dC or transfected with miRNA mimics for 72 h and then seeded into 8-chamber culture slides (Falcon). After two additional days, cells were rinsed with PBS and fixed with 4% paraformaldehyde at RT for 30 min. After treatment with 0.1 M glycine and permeabilisation with 0.1% Triton X-100, cells were subjected to immunofluorescence staining with the anti-MyHC (Millipore) antibody for 1 h 30 min at RT. Cells were washed with cold PBS three times and incubated with Texas Red-anti-mouse secondary antibody (1:100, Jackson Laboratories) at RT for 30 min. The actin cytoskeleton was visualized with TRITC-phalloidin (1:50, Sigma) at RT for 1 h 30 min. Nuclei were counter-stained with 1 μg/ml Hoechst (Sigma). Labelled sections were examined and analysed by using a Zeiss ApoTom epifluorescent microscope (Carl Zeiss) and Axio-Vision software. Experiments were replicated twice.