Mayer hematoxylin solution

Paraffin-embedded tissues were sliced for staining. Paraffin wax sections were dewaxed and then incubated with 0.3% hydrogen peroxide at room temperature for 5–10 min to eliminate endogenous catalase activity. This was then rinsed with distilled water, nonspecific proteins were blocked with 5% goat serum (Solarbio Life Science, Beijing, China), the serum was discarded after 10 min, and not washed. An antibody was added and left at 4 °C overnight, with incubation of paraffin sections with ABC peroxidase and diaminobenzidine (ZSGB-Bio Ltd., Beijing, China) for nuclear staining using Mayer hematoxylin solution (Solarbio Life Science, Beijing, China) counterstainer. For hematoxylin and eosin (H&E) staining, the slides were counterstained using the H&E Kit (Solarbio Life Science, Beijing, China) after they were nuclear stained. Images were acquired using an inverted microscope (Olympus, Tokyo, Japan).

Paraffin-embedded tissues used for H&E staining and IHC analysis were prepared as previously described [30 (link), 31 (link)]. Glioma tissues and mouse brain tissues were incubated with primary antibodies (TMEM158, 1:100, ab98335, Abcam, USA; p-STAT3, 1:100, 9145S, Cell Signaling Technology, USA; and Ki-67, 1:100, 9027S, Cell Signaling Technology, USA), IHC markers were detected using a goat anti-rabbit IgG two-step detection kit (PV-9000, ZSGB-Bio, China). Next, the slides were counterstained with Mayer Hematoxylin Solution (G1080, Solarbio, China) for nuclear staining. The IHC staining images were acquired a VANOX microscope (Olympus, Japan). The intensity score was graded as 0 (negative), 1 (weakly positive, light brown), 2 (moderately positive, brown), or 3 (strongly positive, dark brown). The quantity score was graded as 0 (negative), 1 (≤25%), 2 (26–50%), 3 (51–75%), or 4 (>75%). The total IHC staining score was calculated by adding the intensity and quantity scores.

Tumor tissues excised from mice were fixed overnight in 10% neutral buffered formalin, dehydrated at a gradient concentration, and embedded in paraffin. The tissues were cut into 4 μm thick and fixed on the silicified glass slide. Subsequently, the IHC was carried out using a streptavidin–peroxidase-conjugated method. Briefly, the slides were deparaffinized, rehydrated, immersed in antigen retrieval solution, boiled at 100°C for 10 min, and incubated with a peroxidase inhibitor for 10 min at room temperature. Next, nonspecific binding was blocked with normal goat serum at room temperature for 1 hr, and incubated overnight at 4°C with primary antibodies. After secondary antibodies were incubated at room temperature for 1 hr, 3,3’-Diaminobenzidine tetrahydrochloride (DAB; ZSGB-BIO, Catalog #ZLI-9017, Beijing, China) and Mayer’ Hematoxylin solution (Solarbio Life Science, Catalog #G1080, Beijing, China) were followed. Microscopic images were obtained under a bright field microscope, and the protein expression was visualized by IHC staining and evaluated using the CellProfiler software. The information relating to the antibodies are summarized in Supplementary Table 2.

The antibiotin protein-biotin method got accustomed to performing immunostaining on paraffin-embedded sections. Slides were dewaxed in xylene, then rehydrated in graded ethanol, then the endogenous peroxidase activity was then quenched with 0.3% hydrogen peroxide (China), and the strong antigen recovery solution was heated to 37°C to recover the antigen. 5% goat serum (Solarbio, China) was used to block nonspecific proteins. Primary antibodies (1:100 dilutions) were used to incubate sections at 4°C overnight, subsequently the appropriate biotinylated secondary antibody was added (1:100 dilutions) (ZSGBBio, China) at 37 °C for 60 minutes. The, slides were then hatched with ABC peroxidase and diaminobenzidine (ZSGBBio, China). Next, the slides were counter-stained for nuclear staining by Mayer hematoxylin solution (Solarbio, China). For going on H&E staining, the slides were deparaffinized and rehydrated. Next, the slides were stained by nuclear staining, subsequently re-staining using HE kit (Solarbio, China). The images were taken with an inverted microscope (Olympus, Japan). The human tissue samples used in this study research has complied with the relevant national and institutional policies.

Tissues were paraffin-embedded and sectioned for immunostaining. Slides were dewaxed in xylene, rehydrated, followed by quenching of endogenous peroxidase activity with 0.3 % hydrogen peroxide and non-specific proteins were blocked with 5 % goat serum (Solarbio, China). Sections were incubated with primary antibody at 4◦ C overnight. Next, slides were incubated with ABC peroxidase and diaminobenzidine (ZSGBBio), followed by nuclear staining with Mayer hematoxylin solution (Solarbio) counterstain. For hematoxylin and eosin (H&E) staining, slides were passed through nuclear staining and subsequently re-stained using the H&E kit (Solarbio). Images were acquired using an inverted microscope (Olympus, Japan).