Goat or donkey serum

As previously described (Tan et al., 2020a (link); Tan et al., 2020c (link)), testes were fixed in Bouin’s fixative for 0.5 h at room temperature, and then cleared using multiple changes of 70% ethanol and kept at 4°C. Tissues were dehydrated through an ethanol series—30%, 50%, 70%, 95%, 100%, and 100%—for 10 min each at room temperature, and cleared with two changes of xylene and embedded in paraffin. Tissue blocks were sectioned at 5 μm. Sections were deparaffinized twice in xylene, followed by serial dilutions of ethanol. Unmasking was performed using a steamer (IHC World). Blocking was performed by incubating with 5% goat or donkey serum (Sigma), depending on the usage of secondary antibodies, for 1 hr at room temperature. The sections were then incubated overnight with the primary antibody (listed in Table S3) at 4°C and incubated with secondary antisera (listed in Table S3) for 1 hr at room temperature. The nuclei were counterstained with DAPI (Vector Laboratories), a coverslip was placed over the sections with mounting medium, and the images were viewed using a Leica DMI4000 B fluorescence microscope. Statistical significance was determined using the chi-square test.

Free-floating sagittal brain sections were blocked and permeabilized with 10% donkey or goat serum (Sigma-Aldrich) in Dulbecco’s PBS (D-PBS) containing 0.1% Triton X-100 (Abcam) for 2 h at room temperature (RT). Primary antibodies were then diluted (see antibody section) in 10% donkey or goat serum in D-PBS and applied to sections overnight at 4 °C with gentle agitation. All Alexa Fluor®-tagged secondary antibodies were used at a dilution of 1:500 at RT for 2 h. The sections were then mounted in 4′,6′-diamidino-2-phenylindole (DAPI) mounting medium (Vector Laboratories). Z-stacks and tilescans were captured with ZEN 2 software using a Zeiss 880 scanning laser confocal microscope using a 40X objective lens. High magnification (100X, 2.5X digital zoom with 40X objective) Tuj1-TDP-43-DAPI co-stained images were captured as single Z-plane images. All other images were obtained through single field 30 µm thick (1 image / 5 µm step) z-stack and z-stack tilescan acquisitions and were processed into maximum intensity projections using ZEN 2 software prior to image quantification performed using the Fiji processing package for ImageJ (National Institutes of Health).

Ocy and Ob were seeded on 8-well chamber slides (Corning) coated with or without type I collagen at a density of 1 × 10⁴ cells per well. Cells were fixed with 4% PFA in PBS for 10 min at room temperature (RT) and gently washed with PBS for 5 min three times. After permeabilization with 0.2% Triton X-100 in PBS at RT for 10 min and washing with PBS for 5 min three times, cells were incubated with 2% BSA/PBS and 2% donkey or goat serum (Sigma-Aldrich) for 30 min at RT. Anti-sclerostin antibody (R&D systems, AF1589) or Anti-keratocan antibody (Abcam, ab128304) was applied for overnight at 4 °C with gentle shaking. After washing with PBS, cells were incubated with Alexa Fluor® 594 donkey anti-goat antibody or Alexa Fluor® 594 goat anti-rabbit antibody (Thermo Fischer) for 1 h at RT. Normal goat or rabbit IgG was used for negative control. F-actin was visualized with Alexa Fluor 488 phalloidin (Invitrogen, A12379). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Fluorescent images were acquired with BZ-X800 microscope (Keyence, Osaka, Japan). The percentages of sclerostin- or keratocan-positive cells were calculated (the number of positive cells divided by the total number of cells) and averaged across five random fields per each well.