Ts2r fl microscope

For histological analysis, isolated femurs from euthanatized mice were fixed with 0.1 M phosphate buffer containing 4% paraformaldehyde (Sigma-Aldrich) at 4 °C for a 3 days. Then they were decalcified in 14.5% EDTA solution (sigma) for 1 weeks at room temperature. Decalcified tissues were rinsed, dehydrated, and paraffin embedded in accordance with general protocols. Paraffinized specimens were sectioned in 4 μm thickness with a microtome. Histological study was performed with Harris hematoxylin (BBC Biochemical, Vernon, WA, USA) and Eosin Y (BBC Biochemical). Images were obtained by inverted Nikon TS2R-FL microscope (Nikon).

The mice were sacrificed, and the brain was prepared as described above. Subsequently, the brain was then sliced into 16-µm-thick coronal sections with a Leica Cryostat (Leica, CM1950). Five percent bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 was used to block the brain sections, which were further incubated at 4 °C overnight with the following primary antibodies: Ly-6G antibody (1:100, BD Pharmingen, 551,459), CD45 antibody (1:100, BD Pharmingen, 550,539), cleaved-caspase 3 antibody (1:100, Millipore Sigma, AB3623), neuronal-specific nuclear protein (NeuN) antibody (1:200, Millipore, MAB377). Following washing with TBST, brain sections were incubated with secondary antibody conjugated to fluorescein (1:250, Thermo Fisher Scientific, A32723 and A-11037) for 1 h at room temperature. Sections were washed with PBS and mounted using Vectashield with DAPI. Fluorescence signals in the peri-lesion cortex were observed and captured by a Nikon Ts2R FL microscope (Nikon, Tokyo, Japan). For quantitative analyses, observers were blinded to the experimental groups. The number of positively stained cells per 0.20 square millimeter (0.5 mm × 0.4 mm; 20 × magnification) was counted in three consecutive selected microscopic fields in the peri-lesion cortex as we previously described [11 ]. The average cell number was calculated for each experimental group.

Fixed construct sections were embedded into paraffin, sectioned, and stained with picrosirus red. Images were taken with polarized light at 10x and 20x to observe collagen fascicle organization. Imaging was performed with a Nikon Ts2R-FL microscope equipped with a polarizer and a NIS color camera.