Hematoxylin

Tumors were removed from sacrificed mice, fixed in 4% paraformaldehyde and paraffin-embedded. Paraffin-embedded samples were sectioned (4 μm) and fixed on glass slides. Epitope retrieval of sections was performed in 10 mmol/L citric acid buffer at pH7.2 heated in a microwave. Slides were subsequently incubated with the primary antibody (rabbit anti-Ki67 1:200 dilutions) at 4°C overnight followed by HRP-conjugated secondary antibody for 1 h at room temperature. Antibodies were detected using the substrate diaminobenzidine (DAB, Beyotime; Shanghai, China), and slides were counter-stained with hematoxylin (Beyotime; Shanghai, China).

The BC and adjacent normal tissues were fixed in 10% formaldehyde, embedded in paraffin and sectioned at a thickness of 3~4 μm. The sections were placed in 3% H₂O₂, dewaxed and dehydrated at room temperature for 20 min to block the endoperoxidase activity. The sections were placed in an 80% power microwave for 5 min at 90 °C for antigen retrieval. The sections were incubated overnight with mouse anti-human GSK3β monoclonal antibody (ANT-404, ProSpec-Tany Techno-Gene Ltd, Rehovot, Israel) at 4 °C and then incubated with goat anti-mouse IgG-horseradish peroxidase (HRP; SE134, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 37 °C for 30 min. Hematoxylin (C0105, Beyotime Biotechnology Co., Shanghai, China) was applied for 30 s to stain the nucleus, and diaminobenzidine (P0202, Beyotime Biotechnology Co.) was used for development. The sections were dehydrated until transparent using hydrochloric acid ethanol, sealed with gum, photographed and observed microscopically. The standards used for evaluating the immunohistochemical results were that if the number of stained cells exceeded 25%, and there were obvious brown or yellow–brown granules observed in the cytoplasm, the specimen was positive for GSK3β. The expression rate was determined by the percentage of positive cases from the total cases.

To investigate the effect of abnormal light stimulation on atheromatous plaque formation in mice, we used Oil Red O staining to determine vulnerable plaque formation as previously described [30 (link)]. The arterial arcades were carefully dissected and fixed in 4% paraformaldehyde for 24 h at room temperature. The fixed samples were rinsed three times, each time for 15 minutes. The aortic segments were dehydrated in 20% sugar solution for 24 h and 30% sucrose solution for 24 h. Subsequently, the samples were embedded in Tissue-Tek OCT compound. Serial sections of 10 μm cross-section were prepared and fixed at 4°C using 70% ethanol solution and staining for 1 min in Oil Red O (Sigma-Aldrich, O0625, USA) to identify atherosclerotic plaques. Oil Red O was dissolved in isopropanol and mixed in 3 : 2 (v/v) (water to Oil Red O) and the plaques were stained for 30 min. Then the samples were carefully washed for three times, each time for 5 minutes and stained for 3~5 min in hematoxylin (Beyotime, C0107, China).

Gradient concentrations of xylene and alcohol were used for paraffin section dewaxing and hydration, and then the sections were heated for antigen retrieval. After cooling to room temperature, the sections were soaked in 3% H₂O₂ solution to inactivate the endogenous peroxidase (only for immunohistochemistry). All sections were treated with 10% goat serum for 30 min to block the antigen and then incubated with primary antibodies at 4°C overnight. The secondary antibody (Zhongshan Biology Co. Ltd, China) corresponding to the primary antibody was incubated at room temperature for 30 min. For immunohistochemistry staining, diaminobenzidine was used for antibody staining and hematoxylin (Beyotime, China) for nuclear staining. For immunofluorescence staining, DAPI was used for nuclear staining. The results were observed and photographed under a microscope (Olympus, Japan).
The primary antibodies used are described as follows: IL-1β (1:200, Abcam, United Kingdom); IL-6 (1:200, Abcam, United Kingdom); IL-8 (1:200, Bioworld, United States); K1 (1:200, Abcam, United Kingdom); K6 (1:200, Thermo Fisher Scientific, United States); TNF-α (1:200, Abcam, United Kingdom); IL-23 (1:200, Abcam, United Kingdom); CCL20 (1:100, Abcam, United Kingdom); IL-22; IL-17 (1:250, Abcam, United Kingdom); IFN-γ (1:200, Abcam, United Kingdom); and BrdU (Abcam, United Kingdom).

Samples were fixed in 4% formalin, paraffin-embedded, and sectioned (4 µm). After de-waxing and rehydration, the sections were incubated with 0.01 M citrate buffer for 20 min at 95 °C for antigen retrieval. Endogenous peroxidase activity and non-specific antigens were blocked with 3% hydrogen peroxide (ZSGB-Bio; Beijing, China) and 10% normal goat serum (ZSGB-Bio) respectively, followed by incubation with primary antibody at 4 °C overnight. Sections were rinsed with phosphate buffered saline (PBS), treated with goat anti-rabbit secondary antibody (ZSGB-Bio), visualized using 3, 3′-diaminobenzidine (DAB, ZSGB-Bio) as substrate, and counterstained with hematoxylin (Beyotime; Haimen, China). Normal mouse serum was used as the negative control. Staining of cancer cells was scored as follows: 0, no staining; 1, weak staining in <50% cells; 2, weak staining in ≥50% cells; 3, strong staining in <50% cells; and 4, strong staining in ≥50% cells. The following primary antibodies (Abcam, Cambridge, UK) were used at the dilutions indicated: CCDC109B (1:200), HIF1α (1:200), Ki-67 (1:500), MMP2 (1:100) and MMP9 (1:200).

The following reagents were purchased from Beijing Bioss Biotechnology: c-fos rabbit anti-mouse polyclonal antibody, ZO-1 rabbit anti-mouse polyclonal antibody, Occludin rabbit anti-mouse polyclonal antibody, myosin light chain kinase (MLCK) rabbit anti-mouse polyclonal antibody, P-glycoprotein (P-gp) rabbit anti-mouse polyclonal antibody, multidrug resistance associated protein 1 (MRP1) rabbit anti-mouse polyclonal antibody, multidrug resistance associated protein 2 (MRP2) rabbit anti-mouse polyclonal antibody, goat anti-rabbit immunohistochemistry kit, and hematoxylin. Other reagents used were as follows: glacial acetic acid, analytical grade (Beijing Chemical Works, Batch No. 20100603); osmic acid, acetone, embedding media, uranyl acetate stain, and lead citrate stain (supplied by the Experimental Research Center); potassium permanganate, analytical grade (Beijing Chemical Works, Batch No. 820308); toluidine blue, analytical grade (Sinopharm Chemical Reagents, Batch No. WC20050120); anhydrous ethanol, analytical grade (Beijing Chemical Works, Batch No. 20110); xylene, analytical grade (Sinopharm Chemical Reagents, Batch No. 20106); neutral gum (Sinopharm Chemical Reagents, Batch No. 20120320); hematoxylin (Beyotime Biotechnology, Batch No. C0105); ethylenediamine tetraacetic acid (EDTA), analytical grade (Beijing Chemical Works, Batch No. 840529).