Cryostat 2800 frigocut e

Kidneys were fixed in 4% paraformaldehyde, embedded in paraffin and then cut into 3-µm-thick sections (Cryostat 2800 Frigocut-E, Leica Instruments), and a standard protocol using xylene and graded ethanol was employed to deparaffinize and rehydrate the tissues. The sections were washed with PBS and treated with blocking buffer containing 50 mM NH₄Cl, 2% BSA, and 0.05% saponin in PBS for 20 min at room temperature. The sections were then incubated overnight at 4°C with primary antibodies of anti-NLRP3 rabbit polyclonal antibody (1:150), anti-NKCC2 rabbit polyclonal antibody (1:150), anti-mPGES1 rabbit polyclonal antibody (1:125). After washing with PBS, the secondary antibody was applied, and the signals were visualized using an ABC kit (Santa Cruz Biotechnology, Santa Cruz, CA).

As described in our previous publication^{47 (link)}. In brief, the paraformaldehyde-fixed kidney was sectioned at 4-μm thickness (Cryostat 2800 Frigocut-E, Leica Instruments). Anti-CD90/thymic antigen 1 mouse monoclonal antibody (MRC OX-7) at 1:100 and Alexa Fluor 568 goat anti-mouse IgG (Invitrogen, Grand Island, NY) at 1:2000 were used to label glomerular MCs. Staining was visualized using a confocal laser-scanning microscope (Zeiss LSM510).

Kidneys were fixed in 4% paraformaldehyde, embedded in paraffin and then cut into 3-μm-thick sections (Cryostat 2800 Frigocut-E, Leica Instruments), and a standard protocol using xylene and graded ethanol was employed to deparaffinize and rehydrate the tissues. The sections were washed with PBS and treated with blocking buffer containing 50 mM NH₄Cl, 2% BSA, and 0.05% saponin in PBS for 20 min at room temperature. The sections were then incubated overnight at 4°C with primary antibodies of anti-NHE3 rabbit polyclonal antibody (1:20), anti-NCC rabbit polyclonal antibody (1:200), and anti-ACE rabbit polyclonal antibody (1:200). After washing with PBS, the secondary antibody was applied, and the signals were visualized using an ABC kit (Santa Cruz Biotechnology, Santa Cruz, CA).