Alexa fluor 488 and 594

Rabbit polyclonal antibodies were raised to unique sequences at the N-terminus and C-terminus of Mirk and affinity purified [12 (link)]. The C-terminal antibody was labeled with Alexa Fluor 594 using the Xenon Rabbit IgG Labeling Kit (Molecular Probes) for use in direct immunofluorescence experiments. Alexa Fluor 488 and 594 (highly cross-adsorbed) secondary antibody conjugates and Alexa Fluor 594 phalloidin were purchased from Molecular Probes. Mirk kinase inhibitors EHT5372, EHT6840 and EHT411 were the gifts of Diaxonhit, Paris, France. Antibodies to myogenin, and actin were purchased from Santa Cruz Biotechnology. Tissue culture reagents were obtained from Mediatech. Other reagents were obtained from Sigma.

Exponentially growing HeLa-S3 cells were plated on coverslips and fixed for 15 min at room temperature in 4% formaldehyde in PBS, 0.1% Triton X-100 in PBS(-), and 0.05% Tween-20 in PBS. Fixed cells were rinsed three times in 1× PBS for 5 min each. To visualize H3K27me3 and RNAPII, cells were blocked in blocking buffer (1× PBS, 5% normal serum, and 0.3% Triton X-100) for 60 min, incubated with anti-H3K27me3 (Cell Signaling Technologies, Danvers, MA, USA) and anti-RNA polymerase II clone STD4H8 (Millipore, Billerica, MA, USA) antibodies, and then incubated with Alexa Fluor 488 and 594 (Molecular Probes, Eugene, OR, USA)-conjugated anti-rabbit/mouse IgG in 1× PBS containing 1% BSA and 0.3% Triton X-100. DNA was stained using Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA), and cells were observed on a FluoView FV10i microscope (Olympus, Tokyo, Japan).

The cryostat sections were immersed in a blocking solution containing 10% goat serum for 30 min and incubated overnight with a primary antibody at 4 °C. Macrophages were labeled using ionized calcium-binding adapter molecule 1 (Iba1), which is specific for microglia/macrophages, and CD11b, which is specific for the monocyte/macrophage lineage. The primary antibodies used in this study were rabbit anti-Iba1 (1:1000; Wako Pure Chemicals, Osaka, Japan) and rat anti-CD11b (1:500; M1/70; BD Biosciences, San Jose, CA, USA). The localization of primary antibodies was visualized using secondary antibodies conjugated with Alexa Fluor 488 and 594 (1:500; Molecular Probes; Invitrogen, Carlsbad, CA, USA). Cell nuclei were counterstained with Hoechst 33,342 (Invitrogen). The negative controls lacked primary antibody labeling. Fluorescent images were acquired using a TCS SP8 microscope (Leica Microsystems, Wetzlar, Germany). In wildtype, ICR mice and Csf1r-deficient mice, Iba1/CD11b double-staining was performed. In Ms4a3^tdT transgenic adult mice, Iba1 staining was performed to visualize the ratio of BM-derived macrophages among the total number of macrophages in the inner ear.

Primary antibodies used are as follows: ant-BANF1 (Abcam: ab88464, monoclonal, Sigma: SAB1404629, monoclonal), anti-HexaHis (Abcam ab1187), anti-FLAG M2 (Sigma, F3165), anti-Histone H3 (Cell Signaling, 9715), anti-nucleolin (Cell Signaling, 12247S) and anti-Emerin (Cell Signaling, 5430S). Fluorescent secondary antibodies used are: Donkey anti-Mouse 800 nm (LiCor; IRDye 800CW 926-32212), Donkey anti-Rabbit (LiCor; IRDye 680LT 926-28023) and Alexa Fluor 488 and 594 (Molecular Probes).

Neuron-astrocyte cocultured samples were fixed with 4% paraformaldehyde phosphate buffer solution (Wako, Japan) to preserve the cytoskeletal structures of cells for 2 h at room temperature. To enhance the permeability of the cell membranes and reduce nonspecific binding of antibodies, we used a blocking solution that contained 0.1% [w/v] Triton X-100 (Sigma-Aldrich, USA) and 2% bovine serum albumin (Sigma-Aldrich, USA) in PBS for 4 h at 4 °C. Next, we stained cells with primary and secondary antibodies for 12 h and 6 h in sequential order at 4 °C. The nuclei were stained with Hoechst 33342 (Molecular Probes, USA) for 30 min at room temperature. We washed the samples three times with PBS for 20 min at room temperature in every step. The primary antibodies that were used were mouse anti-β-tubulin III (Tuj-1; Sigma-Aldrich, USA) and chicken glia fibrillary acidic protein (GFAP; Merck Millipore, USA). Alexa Fluor conjugates (Alexa Fluor 488 and 594; Molecular Probes, USA) were used as secondary antibodies. We obtained fluorescent images of cocultured samples by using an inverted confocal laser scanning microscope (LSM 700; Carl Zeiss, Germany) with solid-state lasers (405, 488, and 555 nm). All immuno-stained images were acquired with a 20× objective (NA 0.3), and they were processed by adjusting fluorescent intensities and merged using ZEN 2012 software (Carl Zeiss, Germany).