Vs200 fluorescence microscope

Tumour specimens from nude mice were fixed overnight in 4% paraformaldehyde and embedded in paraffin. The samples were incubated with primary antibodies against cytochrome c (1:100, ABCAM) and P53(1:100, ABCAM) overnight at 4 °C. The samples were then incubated with a secondary anti-rabbit antibody (ABCAM) for 2 h at ambient temperature. Samples were visualised with an Olympus VS200 fluorescence microscope.

Wound tissues were sampled on days 3 and 17 after treatment, embedded in an optimal cutting temperature compound, frozen, and sliced into 10-µm-thick sections at − 22 °C. Sections were then treated with primary antibodies against rabbit anti-CD31 (Abcam) overnight at 4 °C, followed by a 50 min treatment with goat anti-rabbit IgG secondary antibody (Abcam) at 37 °C and 10 min treatment with 4′,6-diamidino-2-phenylindole (DAPI). The stained slides were observed under an Olympus VS200 fluorescence microscope (Japan). To visualize the migration of BMSCs and regenerated nerves in vivo, tissue sections on day 3 and 17 were stained with antibodies against CD90 (ProteinTech), nestin (ProteinTech), and β3-tubulin (Cell Signaling Technology). CD90, nestin, and β3-tubulin signals were visualized using FITC- and CY3-conjugated secondary antibodies (Thermo Pierce). Nuclei were stained with DAPI. Using a laser scanning confocal microscope (VS200, Olympus), images of the sections were obtained for three randomly selected areas for the quantification of fluorescence intensity. All images were post-processed and quantified using ImageJ software.

Immunofluorescence staining was performed on 3 µm paraffin-embedded sections of the kidneys and spleens from 14-week-old lupus mice. The sections underwent dewaxing and hydration steps, followed by heat-mediated antigen retrieval using either sodium citrate (10 mM, pH 6; Catalog No. P0083, Beyotime, Shanghai, China) or Tris–EDTA (10 mM Tris, 1 mM EDTA, pH 9; Catalog No. C1038, Solarbio, Beijing, China, G1480) via microwave for 20 min. To block nonspecific antigens, 5% bovine serum albumin (BSA) was used. Primary antibodies were incubated overnight at 4 °C, while fluorescent secondary antibodies were incubated for 1 h at room temperature. A diluted DAPI solution (1:3 dilution; Catalog No. C1005, Beyotime, Shanghai, China) was applied for 10 min to stain the cell nuclei. After washing with PBS, the sections were sealed using anti-fluorescence quenching sealed tablets. The primary antibodies, fluorescent primary antibodies, and secondary antibodies used for immunofluorescence staining can be found in Additional file 2: Table S1. Immunofluorescence images of the kidneys were acquired using a laser scanning confocal microscope (Leica TCS SP5) and analyzed with Leica LAX software (Leica, Wetzlar, Germany). Immunofluorescence images of spleen T cells and B cells were obtained using an Olympus VS200 fluorescence microscope and analyzed with OlympusVIA software (Olympus, Japan).