Coralite488 conjugated goat anti mouse igg h l

After fixing with 4% paraformaldehyde (in PBS) at 25 °C for 15 min, cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min and blocked with 1% BSA (in PBS) for 1 h. Then, cell samples were incubated with mouse anti-myosin heavy chain 3 (MYH3) antibody (Santa Cruz Biotechnology, CA, USA) or rabbit anti-Ki67 antibody (Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight and, subsequently, with CoraLite488-conjugated goat anti-mouse IgG (H + L) or CoraLite488-conjugated goat anti-rabbit IgG (H + L) (Proteintech Group, Wuhan, China) at 25 °C for 1 h. After staining with DAPI (Sigma-Aldrich, Burlington, MA, USA) for 10 min, samples were imaged with a fluorescence microscope (Mshot, Guangzhou, China). The fusion index was calculated as the percentage of nuclei in MYH3-positive myotubes (≥2 nuclei) to total nuclei within MYH3-positive myotubes [24 (link)]. At least 3 field images were analyzed per sample.
To visualize F-actin in cells cultured in three-dimensional (3D) hydrogels, the samples were first incubated with CoraLite^®488-Phalloidin (Proteintech Group, PF00001) at 4 °C for 2 h. Then, the samples were stained with Hoechst 33342 (Beyotime, C1022) for 10 min and observed using a fluorescence microscope (Mshot, Guangzhou, China).

Indirect immunofluorescence double staining method was performed to visualize TRAF5 and SM22 expression in ASO tissue frozen section using the primary antibodies rabbit TRAF5 monoclonal antibody (1:50, ab303522, Abcam, Cambridge, UK), mouse SM22 monoclonal antibody (1:3000, 60,213–1-Ig; Proteintech Group, Inc., Wuhan, China). We permeabilized the sections with Triton X-100 (0.1%) for 30 min at 4 °C, washed with PBS(1X) and blocked with 5% Fetal bovine serum in PBS for 1 h. The primary antibodies were added to the blocking solution and incubated at 4 °C overnight on an orbital shaker. Next day, following three washings with PBS, the sections were incubated in blocking solution containing the secondary antibodies [CoraLite594 – conjugated Goat Anti-Rabbit IgG(H + L) (1:100; SA00013-4; Proteintech Group, Inc., Wuhan, China) and CoraLite488-conjugated Goat Anti-Mouse IgG(H + L) (1:100, SA00013-1; Proteintech Group, Inc., Wuhan, China)] for 1 h at room temperature. After final washes with PBS three times, we used DAPI to stain the nucleus for 10 min and finally put the anti-fluorescence attenuation sealant( MIKX, DB255, Shenzhen, China) onto a glass slide and cover with a cover glass slip. The sections were examined using a DMi8 microscope (Leica Microsystems).

After the measurement of hearing, mice were sacrificed and dislodged the encapsulated cochlea carefully. Dissected inner ears were fixed with 4% paraformaldehyde, decalcified in EDTA decalcifying solution, microdissected the cochlear epithelium, and then flatten the specimen to orient the sensory hair cell surface side up. Sections were blocked for 1 h with 10% goat serum. The primary antibody, anti-Myosin VIIa (1:50, Abcam) and 4-HNE (1:25, Abcam), anti-GLUT1 (1:500, Abcam), anti-LDHA (1:250, Proteintech), or anti-HIF-1α (1:100 Proteintech), were incubated overnight. Next day, sections were incubated with secondary antibodies for 1 h at room temperature: CoraLite594-conjugated goat anti-rabbit IgG(H+L) (1:250, Proteintech), CoraLite488-conjugated goat anti-mouse IgG(H+L) (1:250, Proteintech). Sections were sealed with mounting medium containing DAPI (Abcam, United States), and photographs were taken by a confocal microscopy (Leica, Italy).

The SHSY-5Y cells were fixed in 4% paraformaldehyde for 10 min at room temperature, followed by three times of PBS washing and subsequent treatment with 10% goat serum albumin (50197Z, Thermo Fisher Scientific, United States) in PBS for 1 hour. The primary antibodies included anti-human NSE (1:150; Cell Signaling Technology, United States) and anti-GAPDH (1:200, 60004-1-Ig, proteintech, China). The secondary antibodies were Cy3–conjugated Affinipure Goat Anti-Rabbit IgG (H + L), (1:100, SA00009-2, proteintech, China), and CoraLite488-conjugated Goat Anti-Mouse IgG (H + L), (1:100, SA00013-1, proteintech, China). SHSY-5Y cell nuclei were stained with DAPI (C1005, Beyotime, China). Images were acquired under a fluorescence microscope.