Alexa fluor 488 goat anti mouse igg antibody

The embryonic dorsal skin tissues were fixed in 4% paraformaldehyde in PBS for 3 h, followed by 15 min wash with PBS and incubated in 30% sucrose overnight at 4°C. Tissues were frozen in OCT (Sakura) and sectioned to obtain 10 µm thickness. The sections were rehydrated and blocked for 2 h in blocking solution (2% goat serum, 1% BSA, 0.1% Triton-X and 0.05% Tween 20 in PBS). cPDSS2 antibody was diluted 1∶100 in blocking solution and incubated overnight at 4°C. The sections were washed three times for 5 min in PBS and incubated with Alexa Fluor 488 Goat Anti-Mouse IgG Antibody (1∶400, Invitrogen) for 2 h at room temperature. The nuclei were stained with DAPI. Samples were analyzed using Olympus Fluoview FV1000 confocal microscope with the same parameters. Images were processed using FV10-ASW and Adobe Photoshop.

Following treatment with 30 nM Ara-C, 100 nM FTD, or 10 μM 5-FU for 6 hours at 37°C, cells were incubated for 15 hours in drug-free medium at 37°C. Cells were collected on a glass slide using Cytospin (Shandon, Pittsburgh, PA, USA). Cells were fixed with 4% formaldehyde for 10 min at room temperature, permeabilized with 0.5% TritonX-100 and, after two rinses in PBS, were blocked in PBS in 5% bovine serum albumin (BSA). The cells were then incubated with anti-γH2AX(Ser139)MAb (Millipore) at a 1/1000 dilution in 5% BSA in PBS for 1 hour at room temperature. After three washes in PBS, the cells were incubated in Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen) at a 1/1000 dilution in 5% BSA in PBS for 1 hour at room temperature, and after they were rinsed in PBS three times, cells were counterstained with 4′, 6′-diamidino-2-phenylindole (DAPI) and mounted with Vectashield (Vecta Laboratories). Cells were observed by the confocal laser scanning microscopy (TCS SP8, Leica Microsystems, Germany). At least fifty cells were scored per data point.

After treating C2C12 myotubes in a 24-well plate with different concentrations of HMGB1 for 72 h, they were washed 3 times with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.5% Triton X-100 at room temperature for 30 min. The primary antibody MyHC antibody was incubated overnight at 4°C, and then incubated with Alexa Fluor 488 goat anti-mouse IgG antibody (1:500; Invitrogen) at room temperature for 1 h, and the cells were stained with DAPI. Images of myotubes were taken by a fluorescence microscope. The diameters of more than 100 myotubes in at least 10 fields were measured by ImageJ software (Zhang et al., 2017a (link)).

N2A cells were plated on top of cover slides in 24-well format and transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were fixed in 4% PFA 24 h post-transfection and immunostained using primary anti-V5 mouse monoclonal antibody (R960-25 Invitrogen) and secondary Alexa Fluor 488 goat anti-mouse IgG antibody (A-11001 Invitrogen). Cells were mounted on microscope slides using ProLong Diamond antifade with DAPI (P36962 Invitrogen) and imaged using a Leica DMI600B microscope.