Rhodamine b

For the preparation of rhodamine B-conjugated alginate, 10 mg of rhodamine B (Invitrogen) was dissolved in 1 mL DPBS, after which 20 mg of 1-ethyl-3-(3(dimethylamino) propyl) carbodiimide (EDC; ThermoFisher Scientific) and 1.75 mg 1-hydroxy-2, 5-pyrrolidinedione (NHS; ThermoFisher Scientific) were added and incubated for 30 min. Ethylenediamine was then added and mixed overnight at room temperature (RT; i.e. 25 ± 1 °C). Following dialysis and lyophilization, rhodamine B-ethylenediamine powder was added to the alginate solution (20 mg alginate dissolved in 2 mL DPBS) and 10 mg of EDC. The solution was stirred at RT overnight and then dialyzed and lyophilized to obtain the final rhodamine B-Alginate powder. Fluorescein isothiocyanate conjugated gelatin (Gelatin-FITC) was purchased from Invitrogen. For the preparation of FITC-conjugated TGF-β2, 30 mg of TGF-β2 was dissolved in 3 mL of 0.1 M sodium bicarbonate buffer, and 15 mg FITC (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 1 mL 0.1 M sodium bicarbonate buffer. The FITC solution was then slowly added to that of TGF-β2 under magnetic stirring. The reaction was incubated overnight at RT with continuous stirring. Finally, the FITC-conjugated TGF-β2 was obtained following dialysis and lyophilization.

To determine the lysosomal pH of different cell lines, an in situ pH calibration curve was established following the reported procedure^{19 (link)}. Firstly, Oregon Green-dextran (Invitrogen) and Rhodamine B-dextran (Sigma) were dissolved in DMEM medium at 5 mg mL⁻¹ at a molar ratio of 1: 1. Cell lines were pulsed with mixed fluorescent dextran for 6 h, chased for 12 h in fresh medium, and washed with PBS to calculate the fluorescence intensity of lysosomes by a confocal microscope (A1R-Storm, Nikon) under 60× oil objective lens. Subsequently, a corresponding calibration was performed for each lysosome. The cells were incubated with nigericin (10 μM) in high-K⁺ buffers from pH 4.0 to 6.0 for 5 min equilibrium on the ice. The fluorescence intensity was measured by the confocal microscope. The Hoechst 33342 (Invitrogen), Oregon Green, and Rhodamine B were excited at 405, 488 and 561 nm, respectively. The ratiometric images were processed by NIS-Elements viewer software, and the resulting quantification was calculated by ImageJ software (NIH), which was plotted as a function of pH value and fitted to a Boltzmann sigmoid to measure the lysosomal pH in various cell lines.

Surgical procedures were performed as previously described^{82 (link)}. Briefly, following a mid-thoracic laminectomy (T9 vertebra), a spinal cord impactor (IH-0400 Impactor, Precision Systems and Instrumentation LLC) was used to induce a contusion injury. The applied force was set to 90 kdyn. Spinal transections were performed following a mid-thoracic laminectomy (T9 vertebra), cutting the spinal cord with spring scissors before filling the void with gel foam. Animal care, including manual bladder voiding, was performed twice daily or as needed following injury.
For dextran-labeling experiments, 1 μL dextran (Rhodamine B, 10,000 MW, ThermoFisher Scientific, Catalog Number D1824) was injected into gel foam separating the transected cord.
Two days after injury, all mice were evaluated in an open field, and all animals exhibiting any hindlimb movements were not further studied. For single nucleus RNA sequencing experiments, a larger cohort of mice was taken through kinematic analysis, and three mice representative of each time point were selected. For histology experiments, at least four mice were used for each condition. Mice with bone-hit contusions or injuries that fell a standard deviation outside of the average behavior for each time point were excluded. Due to animal care requirements injury experiments were not performed blinded.

Soybean oil (SO) was purchased from a local grocery store. Pea protein isolate was obtained from ADM (Decatur, IL, USA). Food grade sodium alginate (Alg) was purchased from Landor Trading Co., Ltd. (Montréal, QC, Canada). Nile red and rhodamine B were purchased from Thermo Fisher Scientific (Waltham, MA USA). Beef brisket fat trimming was cut from a fresh carcass and stored at −18 °C before use.

BSA was purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and appropriate stocks were created prior to use. THPC was purchased from Sigma-Aldrich (St. Louis, Missouri, USA), dilutions were stored at room temperature (RT). Trypsin-Ethylenediaminetetraacetic acid (EDTA) (0.05% (w/v)), 2-propanol, D-mannose, L-fucose, D-galactose were purchased from Sigma-Aldrich (St. Louis, Missouri, USA), Trypan blue, Dulbecco modified eagle medium (DMEM), Phosphate Buffered Saline (PBS), Penicillin-Streptomycin (1000 U/mL), and non-essential amino acid solution (MEM) were purchased from life technologies (Carlsbad, California, USA), Hoechst fluorescent dye (H1399), Rhodamine B, SytoxBlue and limulus amoebocyte lysate (LAL) detection kit were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA) and formaldehyde from Carl Roth (Carl Roth GmbH und Co. KG, Karlsruhe, Germany).

Microneedles were created using a Micropipette Puller PC-10 (Narishige, Tokyo, Japan) to stretch a glass tube with a core (GD-1; Narishige). Using a micro grinder EG-400 (Narishige), the tip diameter was thinned to about 1 μm, and the needle tip was sharpened to an acute angle of 20°. Collected fertilized eggs were carefully arranged in the grooves of a 1.5% agarose hardened in a 15-cm diameter plastic Petri dish under a stereomicroscope and filled with filtered seawater. Next, 200 mM KCl solution containing 0.05% (w/v) dextran and rhodamine B (Thermo Fisher Scientific) was injected, and localization of the dye at each developmental stage was investigated. Images of bright-field eggs were captured using a Nikon DS-Fi2 camera (Nikon, Tokyo, Japan) mounted on a stereomicroscope SZX7 (Olympus, Tokyo, Japan). Fluorescence images were obtained using a LSM-700 confocal laser-scanning microscope (Carl Zeiss, Jena, Germany).