G1005 2

After preparation of mouse spleen slices, samples were stained with hematoxylin staining solution (Servicebio, G1005-1) for 3 to 5 min, washed with tap water, and differentiated with differentiation solution (Servicebio, G1005-3). Subsequently, the slices were washed with tap water and returned to blue with blue return solution (Servicebio, G1005-4). Next, slices were dehydrated in 85 and 95% gradient alcohol for 5 min each and stained in eosin staining solution (Servicebio, G1005-2) for 5 min. Last, slices were placed for 5 min each into anhydrous ethanol I, anhydrous ethanol II, anhydrous ethanol III, xylene I, and xylene II transparent; then, the slices were sealed with neutral gum. Image acquisition and analysis used Panoramic slice scanner and Fiji ImageJ software, respectively.

On the 28th day after CCI operation, three rats in each group (i.e., Sham, CCI, and CCI-Swim groups) were killed, and the spinal cord was collected for histopathological analysis. The spinal cord was fixed in 4% paraformaldehyde solution at 4°C for 90 min. After phosphate-buffered saline (PBS) cleaning for 15 min, the samples were then respectively soaked in 15 and 30% sucrose solution for 24 and 48 h. The tissue samples were placed in compound compounds (SAKURA, 4583), sliced (14 μm thick), and dyed with hematoxylin and eosin (HE). The slices were rinsed with PBS for 5 min, soaked in hematoxylin staining solution (Servicebio, G1005-1) for 4 min, and then washed under running water for 20 min. After washing, 1% hydrochloric acid solution was used for differentiation for 3 s. Rinsing was repeated for 15 min. The slides were kept in 0.1% eosin staining solution (Servicebio, G1005-2) for 3 min and then soaked in 85, 95, and 100% alcohol solutions for 1 min in sequence. After soaking in xylene I and xylene II for 1 min, the tissue samples were sealed with neutral resin and then observed under an Olympus BX53 microscope (Tokyo, Japan).

The histopathological examination of heart tissues was performed using HE staining. Heart samples were put in a 10% formaldehyde solution, dehydrated in an ethanol gradient, embedded in paraffin, and cut into 4-μm sections. After deparaffinization, the sections were stained with hematoxylin (G1005-1; Servicebio, China) and eosin (G1005-2; Servicebio, China) and then mounted and observed under a microscope (Olympus, Japan).