3 3 diaminobenzidine tetrahydrochloride

Paraffin sections were dewaxed with a series of ethanol solutions (100%–70%) and washed again with distilled water and PBS. Antigen retrieval was conducted by heating at 70°C in an EDTA buffer pH 9 for 20 min. After cooling to room temperature, the sections were washed in a PBS solution three times and incubated in 3% hydrogen peroxide for 20 min to remove endogenous peroxidase. Next, 10% goat serum (ZSGB-BIO, China) was used for 20 min to avoid any non-specific reaction. The primary antibodies were incubated at 4°C overnight. Specimens were incubated with biotin-conjugated IgG and horseradish peroxidase-conjugated streptavidin (ZSGB-BIO, China) for 20 min, respectively. The sections were then visualized using 3, 3′-diaminobenzidine-tetrahydrochloride (ZSGB-BIO, China) and washed in distilled water. Finally, hematoxylin staining was added for nuclear staining. All sections were observed under a light microscope (Olympus, Japan).
The following antibodies were used in our study: CD163 (1:500, Abcam, United States), SLC12A2 (1:100, Proteintech, China), ST8SIA1 (1:100, Proteintech), CD24 (1: 50, Santa Cruz, United States), WISP1 (1:100, Proteintech), CD146/MCAM (1: 250, Abcam), and CD90 (1:200, Abcam).

After deparaffinization using xylene and hydration in gradient ethanols, the tissue sections were treated with 3% H₂O₂ for 10 min at room temperature to inhibit endogenous peroxidase activities and then incubated with primary antibodies against substance P (diluted 1:200, Abcam, Cambridge, UK) and HIF-1α (diluted 1:200, Abcam) overnight at 4 °C. The method was the same as our previously described research. After washing with 0.01 M PBS, the sections were incubated with polymer auxiliary agent for 15 min at 37 °C, washed with 0.01 M PBS 5 min × 3 times, and then incubated with Poly-HRP secondary antibody goat anti-mouse/goat anti-rabbit IgG (ZSBio, Beijing, China) for 15 min at 37 °C. After three washes in 0.01 M PBS for 3 min each, the sections were visualized with 3,3-diaminobenzidine tetrahydrochloride (ZSBio) as recommended by the manufacturer. The negative control used 0.01 M PBS instead of antibodies. The sections were examined and photographed with a light microscope (OLYMPUS CX-71, Japan).

Paraffin embedded sections were deparaffinised with Histoclear and rehydrated through graded ethanols. Heat induced antigen retrieval was performed in a microwave oven using Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, 0.05 % Tween 20, pH 9.0) for 20 min and the slides were allowed to cool down prior to blocking with 3 % H₂O₂. After blocking with 5 % (v/v) goat serum in phosphate buffered saline (PBS), sections were incubated overnight at 4 °C with the primary antibodies. After a PBS wash, the sections were incubated with secondary antibody at 37 °C for 30 min. Visualization of a positive reaction was by means of a peroxidase substrate solution containing 0.02 % (wt/Vol) H₂O₂ and 0.1 % (wt/Vol) 3,3′-diaminobenzidine tetrahydrochloride (ZSBIO, Beijing, China) in PBS to produce a brown color, then the sections were counterstained with hematoxylin. A negative control, with the primary antibody replaced by mouse IgG antibody, was always included. The primary antibodies used were anti-proliferating cell nuclear antigen antibody (PCNA) (Merck, Millipore, Darmstadt, Germany) diluted 1:400 to identify cell proliferation. PCNA positive cells were counted in 10 randomly selected × 200 high-power fields under a microscope (OLYMPUS FSX100). The PCNA index was calculated according to the following formula: number of PCNA positive cells/total cell count × 100 % [15 (link)].