Vectashield mounting media with dapi

One-half of each kidney was placed in OCT media and snap-frozen in a slurry containing dry ice and 2-methylbutane (Fisher Scientific, Hampton, NH, USA). Frozen kidney sections were cut into 3 μM sections and stained with goat anti-mouse IgG conjugated to FITC (Pierce) diluted 1:100 or goat anti-mouse C3-FITC (Pierce) diluted 1:100. Kidney sections were fixed in acetone for 10 minutes and washed 3 times with 1X PBS for 5 minutes each. The sections were then incubated with C3 or IgG antibodies in a humid chamber for 1 hour. Slides were mounted using Vectashield mounting media with DAPI (Vector Labs, Burlingame, CA, USA) and examined by fluorescent microscopy. Sections were scored (0 – 4) for immune complex deposition by a pathologist in a blinded manner.

Histological analysis and immunocytochemistry were applied as previously described (Murtha et al., 2022 (link)). Briefly, cochleae from neonatal mice were flushed with 4% PFA and then fixative overnight at 4°C, followed by 3 washes in PBS, then blocked in 5% normal horse serum (NHS) for 1 h at room temperature. Samples were stained with antibodies to OCM (Santa Cruz Biotechnology, USA, sc-7446, 1:200), P2X2 (Alomone Labs, Israel, APR-003, 1:400), the C-terminal-binding protein 2 (CtBP2, BD Biosciences #612044, 1:200) and peripherin (Sigma, USA, AB1530, 1:200). Primary antibodies were incubated overnight at 37°C. Appropriate Alexa Fluor (Thermo Scientific, USA, 1:200) and Northern Lights (R&D Systems 1:200) conjugated secondary antibodies were incubated for 1 hour at 37°C. Slides were prepared using Vectashield mounting media with DAPI (Vector Laboratories, USA). Images were acquired using the LSM800 microscope (Zeiss, Germany) using a high-resolution, oil-immersion objective (Plan-Apochromat 63x, 1.4 NA). Cohorts of samples were immunostained at the same time and imaged under the same optical conditions to allow for direct comparison.