Thy1 yfp transgenic mice

All procedures were conducted in accordance with the National Institutes of Health guidelines and approved by the Institutional Care and Use of Laboratory Animals Committee at the University of California, Riverside (UCR) and University of California, Los Angeles (UCLA). C57BL/6J wild‐type mice and Thy1‐YFP transgenic mice were purchased from the Jackson Laboratory. B6.Cg‐Tg(Thy1‐YFP)16Jrs/J mice (JAX #003709) were backcrossed to wild‐type C57BL/6 mice for more than five generations (41). PLP‐EGFP breeding pairs backcrossed to C57BL/6 mice, a kind gift provided by Dr. Wendy Macklin (University of Colorado, Denver, CO, USA), were bred and housed at UCLA and UCR vivarium facilities. The generation, characterization, and genotyping of these mice has previously been reported (42). Mice were all kept on a 12‐hour light/dark cycle with unrestricted access to food and water.

C57BL/6 wild-type (WT), Thy-1-YFP transgenic mice (003782; The Jackson Laboratory, Bar Harbor, ME), and NLRP3^−/− mice (021302; The Jackson Laboratory) of either sex were used in this study. Thy-1-YFP H line, NLRP3^−/− mice were genotyped by polymerase chain reaction using forward primers to detect mutant: 5′-TGCCTGCTCTTTACTGAAGG-3′, and wide type: 5′-TCAGTTTCCTTGGCTACCAGA-3′; and the common reverse primer: 5′-TTCCATTACAGTCACTCCAGATGT-3′29 (link). NLRP3^−/− mice (2–6-months old) were crossed with Thy-1-YFP mice, which had a small number of RGC labeled30 (link)31 (link), permit visualization of RGCs in vivo. All animal procedures conformed to the guidelines on the Use of Animals from the NIH and the Society for Neuroscience and were approved by the Northwestern University Institutional Animal Care and Use Committee.

The tendon of the adult Lewis rat was donated by Prof. Park, Jong-Woong (Department of Orthopedic Surgery, Anam Hospital). Tail tendons were obtained from Lewis male rats (12 weeks old) purchased from Orient Bio, Inc. C57BL/6 male mice (P3–8 weeks old) were purchased from Orient Bio Inc. Thy1-YFP transgenic mice were obtained from the Jackson Laboratory. The mice were transcardially perfused with 4% paraformaldehyde (PFA) in 1 × Phosphate-buffered saline (PBS), and organs were isolated and post-fixed in 4% PFA overnight at 4 °C. The back parts containing the vertebrae and surrounding muscles were isolated and processed for de-calcification using a solution containing 14% Ethylenediaminetetraacetic acid (EDTA) in ammonium water (pH = 7.2) for 3 days at 4°C^{43 (link)}. Next, the specimens were extensively washed with 1 × PBS.