U pot

Frozen OCT tissue sections were fixed in 4% PFA for 10 min at RT, and then washed three times with PBS for 5 min each. Tissues were then stained with 0.1% picrosirius red (Direct Red 80, Sigma-Aldrich, 365,548 and picric acid solution, Sigma-Aldrich, P6744) for 1 hr and counterstained with Weigert’s hematoxylin (Thermo Scientific, 88,028 and 88029) for 10 min at RT. Polarized light images were acquired using an Olympus IX81 microscope fitted with an analyser (U-ANT) and a polarizer (U-POT, Olympus) oriented parallel and orthogonal to each other. Images were quantified using an ImageJ macro to determine percentage area coverage per field of view using one to five fields of view per tissue region. The ImageJ macro is available at https://github.com/northcottj/picrosirius-red.

The medial menisci were photographed using a Leica M165 FC stereoscopic microscope (Leica). For the histological examination, the medial menisci were fixed in 4% paraformaldehyde for 7 days, decalcified in a 20% EDTA solution for 21 days. We separated the meniscus into three regions—anterior, middle, and posterior—using a scalpel, followed by separating the posterior region of the meniscus into three pieces. We embedded the middle piece of the posterior region in paraffin wax. The area that we selected was entirely within the posterior half of the meniscus that we punctured. The specimens were sectioned along the radial plane at 5 μm. Sections were stained with safranin‐o and fast green, with hematoxylin and eosin, and with picrosirius red (Picrosirius Red Stain Kit; Polysciences, Inc.). Before any staining, the sections were observed using polarizer (U‐POT; Olympus Corp.) and analyzer (U‐ANT; Olympus) for transmitted light microscopy (Olympus BX53). Histological pictures were taken via light microscopy. The medial menisci were evaluated using a histological scoring system for safranin‐o staining, picrosirius red staining, and hematoxylin and eosin staining (Table S4).¹⁰ The scoring was performed by two orthopedic surgeons in a blind manner.

Agxt1-edited mice were subjected to an ethylene glycol challenge, as an overloading of oxalate is needed to develop renal damage and nephrocalcinosis.^{21 (link),22 (link)} The precursor of glyoxylate metabolism ethylene glycol (0.5% Ethylene glycol Reagent Plus; Sigma-Aldrich) was administered in drinking water for 7 days. Hematoxylin and eosin staining was conducted in kidney sections, and analysis was performed using an Olympus BX41 microscope and an analyzer (U-ant) and a polarizer (U-pot) set to detect crystal deposition (Olympus). AGXT protein reduction was verified by Western blot analysis. Liver homogenates were used,^{23 (link)} and protein was detected using an anti-AGXT antibody (1/5000 dilution; ab178699; Abcam). Vinculin (ab129002; Abcam) was used as a load control. Nonedited littermates were used as controls for Western blot analysis and crystal deposition characterization.