Tissue plus

Proximal colon was harvested and flushed with ice cold PBS prior to fixation in 4% PFA at 4°C for 18 h. After washing in PBS and cryoprotection in PBS containing 30% sucrose, colon samples were embedded in OCT (Tissue Plus, Fisher Scientific 23-730-571) and cryosectioned at 16 μm. Sections were washed in PBST (0.3% Triton X-100) and blocked in 3% normal donkey serum PBST for 2 h at room temperature. Sections were incubated in primary antibodies (1:500) to GFP (ab13970,), β-tubulin III (Biolegend 802001), or α-SMA (Sigma A2547) prepared in blocking buffer at 4°C for 18 h. Following three 20-min washes in PBST, sections were incubated in fluorescent-conjugated secondary antibodies (1:800) purchased from Jackson ImmunoResearch for 1 h at room temperature along with Hoechst 33342 for cell nuclei visualization. After washing with PBST, the sections were coverslipped with Fluoro-gel (EMS). Tissues from Tg(Opn3-eGFP)JY3Gsat (MMRRC stock number 030727-UCD) and GFP-negative control C57BL/6 mice embedded in the same OCT block and processed in parallel. Images were captured with Nikon Eclipse Ti2 confocal microscope.

Mice were euthanized with exposure to CO₂ followed by cervical dislocation, and all the implants were detached before MicroCT scanning 6 weeks after the primary surgery. Femurs of operated sides were then removed, and femurs were fixed in 4% paraformaldehyde overnight and decalcified in 0.5 M ethylenediamine-tetra-acetic acid (EDTA, pH 7.4) for 2 weeks. The specimens were then placed in embedding medium (optimal cutting temperature or OCT) (Tissue Plus^®, Fisher HealthCare, Hampton, NH) for frozen specimens. Finally, the region of interest located 3 mm from the distal end of femur was cut into transverse sections with 10 μm-thickness for subsequent staining (Lin et al., 2016 (link)).

Animals were deeply anesthetized and then transcardially perfused using a peristaltic pump with phosphate buffered saline (PBS) followed by chilled 10% formalin (Fisher Scientific). Brains were extracted from the skull after perfusion, postfixed overnight at 4 °C, cryoprotected in 30% sucrose in PBS, embedded in O.C.T. compound 4585 (Tissue-Plus, Fisher HealthCare) and stored at −80 °C until sectioning. 50-μm thick sagittal sections were cut with a Leica CM3050 S cryostat. To remove the cryoprotective solution, sections were washed with PBS. Sections were mounted on slides and covered with Fluoroshield anti-fade reagent with DAPI (Sigma). Images were acquired on an inverted fluorescent microscope (Nikon Eclipse Ti) using NIS-Elements AR software. Image processing was performed in Python.

Eyes with retinal detachment were stained with terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) apoptosis kit as previously described [17 (link)]. Briefly, detached retinas were carefully enucleated and embedded in O.C.T compound (Tissue-Plus; Fisher Scientific, Waltham, MA, USA) with the detach regions facing down. Non-consecutive serial sections were captured at 10 µm thickness on a cryostat (CM1950; Lecia) at the peak of the detached region approximately 800–1000 µm from the self-sealing wound. Detached sections were stained with a TUNEL apoptosis kit following the manufacture’s protocol (EMD Millipore, Burlington, VT, USA) and stained with DAPI nuclear dye. The ONL layer of adjacent sides of the detached retina were imaged with an epifluorescence microscope (Axio Observer Z1; Zeiss Oberkochen, Germany) using a 40X lens. TUNEL positive cells were manually counted using the cell counter function on ImageJ for four sections per detached retina. The ONL thickness was measured by manually outlining using ImageJ freehand tool for at least eight regions per retina. The total representative TUNEL positive cells per mm² was calculated by dividing the averaged TUNEL positive cells per averaged ONL area.

Depending on the period of treatment, animals were sacrificed at PD47, PD91 and end-stage (humane endpoint). Mice were anesthetized and perfused with 0.01M phosphate buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde (PFA) for tissue fixation. Spinal cords and hindlimb gastrocnemius muscles were dissected. Then, spinal cords were post fixed overnight at 4 °C and gastrocnemius muscle tissue was post fixed for 30 min at room temperature using the same fixative. Next, all the tissues were transferred to 30% sucrose solution for cryopreservation. Then, lumber spinal cord segments 1–3 (about 0.5 mm) and right medial gastrocnemius muscle were embedded in Optimal Cutting Temperature (OCT) compound (Tissue Plus, Fisher Healthcare, Houston, TX, USA) and serially cryosectioned at 20 µm by a cryostat (Leica, Wetzlar, Germany).

AR113Q knock-in mice were generated using exon 1-specific gene targeting^{50 (link),59 (link)}, backcrossed to C56BL/6 (≥10 generations), and housed in a specific pathogen-free facility on a 12-h light/dark cycle with chow and water ad libitum. For analysis, AR113Q males were used at 3 months of age and randomly assigned to treatment groups. JG-294 was dissolved in 70% propylene glycol (W294004, Sigma-Aldrich) and 30% PBS (pH 7.4, Life Technologies), heated to 95 °C to dissolve compound, aliquoted and frozen. Mice received intraperitoneal injections (3 mg kg⁻¹) every other day for 2 weeks. 17-DMAG (S1142, Selleckchem) was dissolved in 1% DMSO (D2650, Sigma-Aldrich), 30% polyethylene glycol (202371; Sigma-Aldrich) and 1% Tween80 (P4780; Sigma-Aldrich), and administered by i.p. injection (10 mg kg⁻¹) every other day for 2 weeks. At the end of treatment, mice were euthanized and quadriceps were harvested and flash frozen in liquid nitrogen or mounted on OCT (Tissue-Plus, Fisher Health Care) for cryosectioning. For western blot, lysates were obtained by homogenizing tissue in RIPA containing phosphatase and protease inhibitors. All procedures involving mice were approved by the University of Michigan Committee on Use and Care of Animals (PRO00008133) and conducted in accordance with institutional and federal ethical guidelines for animal testing and research.