Alexa fluor 568 conjugated goat anti mouse igg h l secondary antibody

Fixed tumorspheres were permeabilized with 0.1% Triton X-100 for 30 min. The cells were then stained with 5 μg/mL of primary antibody overnight at 4 °C and 1 μg/mL of Alexa Fluor 568-conjugated goat anti-mouse IgG (H + L) secondary antibody (ThermoFisher) at room temperature for 1 h. Z-stack confocal images (10×) were captured and analyzed using high-content imaging. Mouse anti-human CD45 primary antibody (CD45-2B11, eBioscience™, San Diego, CA, USA), FITC-conjugated CD3 antibody (UCHT1, eBioscience™), mouse anti-human CD8a antibody (RPA-T8, eBioscience™, San Diego, CA, USA) and PE CF594-conjugated CD163 (GHI/61, BD) were used in the experiments.

The kidneys were fixed with 4% paraformaldehyde in PBS for 16 hours at 4°C, sequentially immersed in 10%, 15% and 20% sucrose in PBS for 12 hours at 4°C, embedded in optimum cutting temperature (OCT) compound and immediately frozen in liquid nitrogen.
The immunofluorescence staining was performed using a primary antibody and a secondary antibody (Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (H+L) secondary antibody or Alexa Fluor^® 568-conjugated goat anti-mouse IgG (H+L) secondary antibody, [Thermo Fisher Scientific]). The sections were blocked for 1 hour and incubated with a 500-fold dilution of the primary antibody for 16 hours at 4°C. After the incubation with the secondary antibody for 1 hour at room temperature in the dark, the sections were incubated with DAPI (4’,6-diamidino-2-phenylindole, dihydrochloride) (Thermo Fisher Scientific) and mounted using VECTASHIELD Mounting Medium (Vector Laboratories). The images of the sections were captured using Axio Observer Z1 and Axio Vision (ZEISS, Tokyo, Japan).