Vectashield antifade mounting medium h 1000

Mouse brain sections were fixed in 2% PFA for 30 min, then permeabilised in PBS/0.5% Triton X-100 for 1 h, blocked in PBS with 1% BSA (blocking solution, BS) for 1 h and incubated overnight at 4 °C with a NeuN antibody (1:2500) in BS. They were subsequently incubated for 1 h with a secondary fluorescently labelled antibody (Alexa488, Alexa568, Alexa647; Thermo Fisher, Waltham, MA, USA) in BS (1:1000). After post-fixation with 2% PFA for 10 min, to obtain access to DNA methyl groups, cell nuclei were mildly depurinised with 4N HCl treatment for 15 min and incubated with antibody-enriched culture medium against mCG or mCA in BS overnight at 4 °C. After incubation for 1 h with a secondary fluorescently labelled antibody in BS (1:1000) the tissue was stained with 1:100 YOYO1 (Thermo Fisher, Waltham, MA, USA) for 15 min and mounted with Vectashield Antifade Mounting Medium (H-1000, Vector Laboratories, Newark, NJ, USA). Washing with PBS/0.05% Tween-20 for 1–2 h was performed after each step, reagents were dissolved in PBS/0.05% Tween-20 and steps performed at room temperature, unless otherwise stated.

RAW 264.7 cells were cultured on a glass coverslip in a 24-well culture dish for 4 days in the presence of sRANKL. The culture medium was changed every 2 days. The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, washed twice with PBS, permeabilized with 0.5% NP-40 (492016, Calbiochem) in PBS for 5 min and incubated with 1% bovine serum albumin (A7906, Sigma-Aldrich) in PBS for 1 h. The cells were then incubated with the primary antibody (non-muscle myosin IIA, cortactin, vinculin, paxillin or zyxin) in 1% bovine serum albumin in PBS at 4°C for 16 h, washed twice with PBS, and incubated with the appropriate secondary antibodies and rhodamine-phalloidin in 0.1% bovine serum albumin in PBS for 30 min. The stained cells were embedded with VECTASHIELD Antifade Mounting Medium (H-1000, Vector Laboratories, Burlingame, CA). Confocal imaging was performed on a Nikon A1si microscope equipped with a 60×/1.3 NA oil objective lens (Nikon, Tokyo, Japan). 3D images of the ZLS were reconstructed from z-section images acquired at 0.3 or 0.4 µm intervals. The nominal optical resolution was 0.3 µm and 0.49 µm along the xy-axis and z-axis, respectively. Nikon NIS-Elements AR-3.0 software was used for image analysis. The images shown were adjusted for contrast and brightness using Adobe Photoshop CS.

Cells were cultured in 4-well chambered slides, fixed in 4% paraformaldehyde and incubated in 1:200 diluted γH2A.X antibody (CST: 9718) overnight at 4 ^oC after blocking as described previously [36 (link)]. 1:200 Alexa Fluor 488 (Molecular Probes, ThermoFisher) was used to collect signals. Cells were counter-stained with 5 μM Hoechst 33342 (Abcam: ab228551) for 5 min and mounted using VECTASHIELD^® Antifade Mounting Medium H-1000 (Vector Laboratories) and visualized under 40x magnification using Zeiss LSM confocal microscope. Five images were clicked randomly using ZEN 2 (blue edition) software and quantification of green fluorescence was normalized with blue using ImageJ software (National Institutes of Health) and plotted as a graph.

Fluorescent images were acquired using a ZEISS LSM 710 Confocor3 laser scanning confocal microscope, with 25× or 40× oil immersion objectives, numerical aperture 0.8 and 1.3, respectively, at 512 × 512- or 1,024 × 1,024-pixel resolution, with 0.51- or 0.96-µm steps, throughout the entire larval VNC. Specimens were fixed and mounted in Vectashield Antifade Mounting Medium (H-1000; Vector Laboratories) at RT. The fluorochromes used were Alexa 488, 546, and 633, directly conjugated to secondary antibodies, and membrane-tethered GFP and YFP, fused to histone. Stacks of images spanning the entire VNC were acquired using ZEN software (ZEISS). Bright-field images were acquired using an Axioplan 2 microscope (ZEISS) using Nomarski optics and an AxioCam camera, with 63× oil immersion objective, numerical aperture of objective 1.4 and oil condenser, adapter 0.63×, and ZEN acquisition software. Stacks of images were processed using ImageJ, and Photoshop and Illustrator (Adobe) were used to process images and compile figure plates.

ASMCs were seeded onto coverslips in 24-well plates and incubated under various conditions for up to 48 h. Afterwards, ASMC were fixed by 4% formaldehyde (15 min, room temperature), washed once with DPBS, permeabilized by 0.15% Triton (15 min., room temperature), and blocked with 5% bovine serum albumin (BSA) in DPBS for 1 h. The primary mouse anti-Cytochrome c (#556432, Thermo Fisher Scientific) antibody or FITC anti-human FcεR-Iα (#334607, BioLegend, San Diego, CA, USA) was stained overnight at 4 °C. Alexa488-labeled anti-mouse secondary antibody (#11001, Thermo Fisher Scientific) was used to visualize Cytochrome c. F-actin is stained with TRIC-Phalloidin. Nuclei were stained with DAPI. Coverslips were mounted with Vectashield Antifade Mounting Medium (H-1000, Vector Laboratories, Burlingame, CA, USA). Images were acquired by using the Nikon Confocal A1 microscope with a 40× 1.3NA FI oil objective of 0.225 μm Z-stack step. The printed images are presented as Z-stack projection generated by imaging software FIJI. Single cell (50 cells/group) cytochrome c expression was calculated by FIJI with the “3D objects counter” plugin and finally normalized to the control group after 6 h. The cellular FcεR-Iα expression level was measured by the “Analyze Particle” feature of FIJI, quantified by an average of 10 cells/cell line, five cell lines/group, and analyzed by one-way ANOVA.