G1100

The histopathology examination of SACC xenograft tumour tissues was performed with haematoxylin–eosin staining. Briefly, after deparaffinization and rehydration, slides were stained with haematoxylin (Maixin Biotech, CTS‐1097) and eosin (Solarbio, G1100). The slides were then observed using an Olympus BX43 microscope (Olympus Corporation).

The eyeballs were fixed in 4% paraformaldehyde for 24 h at room temperature, dehydrated with 30% sucrose before transferred into OCT medium (4583, SAKURA) and then frozen at −20 °C. The tissues were sectioned into 8 μm in thickness and placed on glass slides. The tissues mounted on slides were then washed with ddH₂O for 10 min to remove OCT. For hematoxylin-eosin staining, slides were stained with hematoxylin (MINDEL GTS-1096) for 5 min, then stained with eosin (G1100, Solarbio) for 90 s. For immunofluorescence staining, slides were treated with 0.5% Triton (V900502-100ML, Sigma) for 15 min followed by 10% donkey serum for 1 h before incubating with primary antibodies overnight at 4 °C. On the next day, sections were washed with 0.1% Tween 20 in PBS (PBST) and incubated with the secondary antibodies for 1 h at room temperature. The nuclei were stained with Hoechst 33324 (H3570, Thermo Fisher Scientific). The antibodies (Abcam) against CD31, α-smooth muscle actin, Ly6G and MPO were used as primary antibodies. Secondary antibodies were Alexa 488-conjugated-goat anti-rabbit IgG (Abcam), Alexa 594-conjugated-goat anti-rat IgG (Abcam), and Alexa 555-conjugated-goat anti-mouse IgG (Thermo Fisher Scientific). Images were captured under a laser-scanning confocal microscope (Leica TCS SP8, Leica).

The lung tissue sections of the mice were dewaxed and subjected to HE staining. First, they were stained with hematoxylin staining solution (Solarbio, Beijing, China, G1140) for 3 min, placed in pure water until they returned to blue, infiltrated with 75% ethanol for 1 min and then 95% ethanol for 5 min, and then stained with eosin staining solution (Solarbio, G1100) for 2.5 min. They were dehydrated using 90% ethanol and xylene. After dyeing, neutral resin was used to seal the slide, the resin was allowed to dry, and images were taken under a microscope (Nikon, Tokyo, Japan, 594200).

Hematoxylin-eosin (H/E) staining was performed as previously described [5 (link)]. Briefly, spinal cord paraffin sections were baked at 60^oC for 2 h and dewaxed and hydrated. Then, the slices were placed into distilled water and hematoxylin aqueous solution for staining for 3 min. Next, slices were rinsed with running water and stained with eosin-lasting solution (G1100; Solar bio) for 3 min. Finally, the slices were dehydrated, transparentized, and sealed. The sections were stained with H/E, and then images were observed and captured with a light microscope (Leica DM2500, Wetzlar, Germany).

Following the behavioral testing, the animals were sacrificed for HE staining. After transcardial perfusion fixation, the brain was rapidly removed and post-fixed for 12 h at 4°C. Paraffin sections (4 μm thickness) were cut using a slicer (SLEE, CUT5062, Germany), mounted onto gelatin-coated slides, air-dried, and stored at room temperature for later use.
For HE staining, sections were dewaxed, rehydrated, and incubated with hematoxylin solution (Solarbio, Cat: H8070, China) at 22–24°C for 15 min. After washing in a hydrochloric-alcohol solution for several seconds, the sections were incubated in an eosin solution (Solarbio, Cat: G1100, China) for 1 min followed by alcohol dehydration, xylene clearing, and coverslipping.

Tissues were processed to obtain paraffin-embedded sections and then analyzed by hematoxylin and eosin staining according to the manufacturer’s protocol (Cat. No. G1100, Solarbio, China). For immunofluorescence analysis, 7 μm sections were incubated with primary antibodies overnight at 4 °C, washed with 0.25% Triton X-100 in PBS, and incubated with fluorescence-labeled secondary antibodies for 2 h. Sections were imaged using a GENERTION2 microscope (Discover Echo Inc., USA).
Cardiomyocytes were rinsed with PBS, fixed with 4% paraformaldehyde for 10 min and then permeabilized with 0.1% Triton X-100 for 10 min. The cells were incubated with rabbit anti-DDX17 antibody for 12 h. The cells were washed three times with PBS for 5 min and then mouse anti-BCL6 antibody was added for 12 h. The cells were then incubated with goat anti-rabbit secondary antibody (red) and goat anti-mouse secondary antibody (green) for 1 h at 37 °C. The nuclei were then counterstained with Hoechst 33342 (Cat. No. 875756-97-1, Merck, USA) or DAPI (Cat. No. D9542, Merck, USA). Cardiomyocytes were then examined by fluorescence microscopy (Olympus BX51, USA). Images were merged using Adobe Photoshop CS6 (Adobe Systems, USA).