Tissue Staining and Microscopic Analysis

Hematoxylin and Eosin (H&E) staining was performed using a previously established protocol for frozen tissues³⁶ . Picrosirius Red staining was performed following manufacturer’s protocol (Abcam). Due to tissue fragility, the thyroid cartilage was removed on sections to produce cleaner stains (i.e., less folds) (Supplementary Fig. 7). CD11b staining was performed using the same protocol and antibody (Mouse Anti-Rabbit CD11b [Bio-Rad MCA802GA], 1:100 dilution) described previously^{15 (link)} Briefly, sections were rehydrated with PBS and incubated with blocking buffer (PBS, 5% BSA, 5% milk, 5% FBS) for 30 min at room temperature. Next, the primary antibody was applied overnight at 4 °C and the slides were washed with PBS before applying the secondary antibody (AlexaFluor 488 Goat Anti-mouse) at a 1:1000 dilution for 1 h at room temperature. Finally, the slides were washed with PBS stained with DAPI, and mounted with Prolong Gold Antifade mountant. H&E and Picrosirius Red brightfield imaging was performed by the University of Virginia Biorepository and Tissue Research Facility (BTRF) core using a Hamatsu Slide scanner. Picrosirius Red polarized light imaging was performed using a LEICA Thunder microscope at the UVA Advanced Microscopy Facility. Polarized light image analysis was conducted using ImgeJ by separating the red and green channels, auto-thresholding each channel, and then measuring the percent area (Supplementary Fig. 12). For this analysis, the new tissue was cropped in each image to analyze the full area and the full right vocal fold was analyzed. Three 10X sections were analyzed for each of the rabbits that had new tissue formation (n = 4). Fiber alignment analysis was conducted using a previously published MATLAB method available on github^{29 (link),32 (link)} with a square size of 100 and threshold of 10000 (Supplementary Fig. 13). Three 20X images from each vocal fold with new tissue were analyzed using the right vocal fold as a comparison. Three different sections were used for each rabbit (n = 4).