G1005

Paraffin-embedded sections (3–4 μm in thickness) of tissue surrounding the lesion site in the spinal cord and spleen were stained using an HE staining kit (Servicebio, G1005), a Nissl staining kit (Servicebio, G1036), and Prussian blue staining kit (Servicebio, G1029). After the addition of neutral gum to the spinal cord slices, the slides were covered, and the sections were observed under a microscope.

The tumor issues were fixed, dehydrated by gradient ethanol and xylene, then immersed in wax. Then, the tissues were cut into 5 μm slices, dewaxed and hydrated by xylene and gradient ethanol, and stained with HE staining (SERVICEBIO, G1005). At last, ethanol with low to high concentrations was added to dehydrate. Vitrification by xylene and the slices were sealed.

The paraffin embedded ovarian tissues were sliced into 4 μm-thick sections, and mounted onto the poly-L-lysinecoated slide, some were stained by hematoxylin and eosin (H&E) according to standard procedures (G1005, Servicebio, Wuhan, China), some were for immunohistochemical (IHC) test according to standard procedure as reagent kit (K5007, Agilent Dako, USA). Briefly, deparaffinized sections were incubated in a sodium citrate buffer (pH 6.0), 10 Mm at boiling temperature for 30 min, blocked in 2% PBS-BSA for 20 min, incubated with rabbit anti-rat HSP70 antibody (ab181606, Abcam,UK) at 4 °C overnight, and the slides were subsequently incubated with labeled dextran polymers goat against rabbit IgG at room temperature for 30 min and staining with 3,3′- diamino-benzidine (DAB) for 3 min of twice and and counterstaining with hematoxylin. For negative control PBS was used as a substitute for the primary antibody. IPP6.0 software was used to analyze the optical density of IHC photographs. Three 400 × photographs of each slide were selected for optical density analysis.

Dissected colon tissues were immediately immersed in 4% paraformaldehyde (Sigma-Aldrich, P6148). The tissue samples were processed in molten paraffin in cassettes and kept at -20°C until paraffin solidified completely. Paraffin blocks were trimmed and then sectioned into 4 μm sections using a microtome (Leica, SP1600). The sections were picked up with a paintbrush, placed on the surface of deionized water in a bath at 40°C, and transferred onto histological slides. The slides were oven-dried (60°C) and dehydrated with different concentrations of alcohol (Sinopharm, 100092683), and samples were stained with hematoxylin solution (Servicebio, G1005) for 5 min and differentiated with acid alcohol (Sinopharm, 100092683). After washing the slides under slowly running tap water, the samples were stained with eosin (Servicebio, G1005) for 5 min, dehydrated with alcohol (Sinopharm, 100092683), and then photographed under a light microscope (Olympus, BX53).

After in vivo therapy and toxicity examination, all the mice were euthanized, the major organs and subcutaneous tumors were removed, washed with PBS and fixed in 4% fixative solution (Cat. no. P1110, Solarbio) and then embedded in paraffin. After that, the paraffin-embedded tumor sections were cut using a microtome (Leica RM2235, Germany) and mounted on slides. Finally, H&E-stained (G1005, Servicebio) tumor sections were stained using standard histological techniques. Anti-Ki-67 (catalogue no. GB111499, Servicebio), anti-Ly6G (catalogue no. GB112299, Servicebio), anti-HIF-1α (catalogue no.36169 T, Cell Signaling Technology), TUNEL (G1510, Servicebio), cleaved caspase-3 (catalogue no. 9661 S, Cell Signaling Technology) were employed for different sections staining, respectively. Finally, slices were photographed with a Virtual slide microscope (Olympus VS120, Japan). For immunofluorescence studies, the images of stained slices were obtained using a confocal laser scanning microscope (Zeiss LSM 710).

Paraffin slices were used to view morphological changes in the intestinal tissue; see Table S2 for details. Fresh tissues were immersed in a 4% paraformaldehyde solution (E672002, Sangon Biotech, Shanghai, China) for seven days, dried, embedded in paraffin, sectioned (4 μm), and stained (hematoxylin and eosin staining) (G1005, Servicebio, Wuhan, China). The M8 automated digital scanning imaging system (Wanbangjunyi, Beijing, China) was used to observe and photograph the paraffin slices. The data were measured using the image-pro plus 6.0 by two observers who were unaware of the study’s design.

After fixing the tumor tissue samples in 10% formalin, and cut into 7-μm slices, the sections were stained with hematoxylin and eosin (G1005, Servicebio, China) for H&E staining. For Ki-67 immunohistochemistry, Anti-Ki-67 antibodies were added to the sections and cultured overnight at 4 °C, followed with incubation with the matched secondary antibody for 2 h. Subsequently, DAB substrate was used to stain the sections. And then, the sections were dehydrated and sealed with coverslips after counterstaining with hematoxylin. For the TUNEL assay, the section staining was performed using a TUNEL kit. After being dewaxed and hydrated, sections were treated with proteinase K solution for 15 min, followed with the treatment of DNaseI reaction solution (100 μL). After incubation with 100 μL of TdT enzyme reaction solution for 1 h at 37 °C in the dark and then incubated with 100 μL of streptavidin-HRP for 30 min at 37 °C, a high-capacity digital slide scanner system was applied to analyze the sections.