Anti rabbit igg alexa fluor 594

Brightfield images were captured using a Nikon ECLIPSE Ti inverted microscope. Whole-mount immunofluorescence staining was conducted for histological analysis. After fixation, samples were washed three times in DPBS, 5 min each time, followed by 20 min permeabilization in 0.3% Triton-X 100 and 2 h blocking in 1% (w/v) bovine serum albumin (BSA) with 0.1% (v/v) Tween 20. For sarcomeric alpha-actinin (SAA) staining, rabbit anti-SAA antibody (Abcam, cat. ab68167) was applied overnight at 4 °C and anti-rabbit IgG Alexa Fluor 594 (Abcam, cat. ab150080) was subsequently applied at room temperature for 2 h.
Cross-sectional images were acquired for cross-sectional area and specific force assessment. After fixation, EMT samples were dehydrated using 30% (w/v) sucrose solution for 30 min, dried on Kimwipes^® (Kimtech Science, cat. 06-666), then embedded in Scigen Tissue-Plus^TM OCT compound (Fisher, cat. 23-730-571) and frozen in dry ice overnight. Embedded EMT samples were then cross-sectioned into 4-μm cryosections near the center, permeabilized in 0.3% Triton-X 100 for 3 min, blocked in 1% (w/v) BSA with 0.1% (v/v) Tween 20, then stained for SAA following the above-mentioned procedure. Images were acquired using Leica SP8 inverted confocal microscope and LAS X image processing software.

Next, 7-μm frozen sections were incubated with anti-CD11c (Invitrogen) and secondary antibody (anti-hamster IgG, Southern Biotech, Birmingham, AL, USA), and sequentially incubated with TSA Fluorescein System (Akoya Biosciences, Marlborough, MA, USA). The sections were also incubated with anti-collagen type 1 (Novotec, Bron, France) and secondary antibody (anti-rabbit IgG Alexa Fluor 594, Abcam). Finally, the sections were incubated with DAPI (Invitrogen). Images were acquired using a BX50 microscope and its imaging system (Olympus, Tokyo, Japan). The positive signal of each colocalized area was selected according to the method of Tolivia et al. (Figure S10) [63 (link)] and analyzed using Adobe Photoshop CS software, version 8.

3T3-L1 cells were grown on 0.2% gelatin-coated coverslips and induced to differentiate into adipocytes with standard DMI. At the indicated time point cells were fixed with 95% ethanol, followed by permeabilization with 0.1% Triton X-100 and 0.5% NP-40 on ice. After blocking with 5% nonfat dried milk in TBST, the coverslips were incubated with primary antibody, namely, mouse anti-METTL3 (Abnova), rabbit anti-METTL14 (Sigma), goat anti-WTAP (Santa Cruz), mouse-anti-SC-35 (Abcam), or mouse anti-Smith antigen (Thermo Fisher Scientific), overnight at 4°C. The coverslips then were incubated with fluorescent dye-conjugated secondary antibody, namely, anti-mouse IgG–Alexa Fluor 488 (Cell Signaling Technology), anti-rabbit IgG–Alexa Fluor 594, or anti-goat IgG–Alexa Fluor 647 (Abcam), for 0.5 h at 37°C and mounted with DAPI-containing medium (Life Technologies). Fluorescent images were acquired using an FV10i confocal microscope (Olympus).