Fv10 asw 2.1 viewer software

Cells plated on coverslips were rinsed twice with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Cells then were washed 3 times with PBS, permeabilized (10 min) in 0.4% Triton X-100 for 10 min, and blocked (1 h at room temperature) in PBS with 0.5% Tween 20 (PBST) containing 4% bovine serum albumin (Sigma). After overnight incubation at 4 °C with the indicated primary antibody, cells were washed with PBS (3 × 10 min) and then incubated (2 h at room temperature) with Alexa Fluro 568 goat anti-rabbit IgG (Invitrogen) or Alexa 568 goat anti-mouse IgG (Invitrogen). After washing with PBS (3 × 10 min), cells were exposed for 5 min to 0.5 μg/ml of DAPI (Sigma). Following final washes with PBS (3 × 10 min), the coverslips were mounted using Fluoromount Aqueous Mounting Medium (Sigma) and imaged using an Olympus Fluoview FV1000 confocal laser scanning microscope. Raw images were analyzed using the Olympus FV10-ASW 2.1 Viewer software (Olympus).

Following fixation with 4% paraformaldehyde (ACMEC) and cryopreservation in 30% sucrose (MACKLIN), the optimal cutting temperature‐embedded kidney tissues were sectioned to 4 μm thickness. Sections were permeabilized with 1% Triton X‐100 (Sigma Aldrich) for 8 min and incubated in a blocking solution (PBS with 1% bovine serum albumin, 0.3% Triton X‐100) for 30 min at room temperature (25°C). Sections were incubated with primary antibodies overnight at 4°C according to the manufacturer's instructions, washed with PBS, and then incubated with the appropriate secondary antibodies in blocking solution for 1 h at room temperature (25°C). After the addition of secondary antibodies, nuclei were stained, and samples were mounted using mounting medium with DAPI (#ab104139; Abcam). All fluorescence images were captured using a confocal FV1000 microscope (Olympus), and images were processed and analyzed using the FV10‐ASW 2.1 Viewer software (Olympus). Primary antibodies included: CD206 (#ab64693; Abcam), F4/80 (#144801, clone BM8; eBioscience), and TGF‐β1 (#ab215715; Abcam).