Sabc kit

Samples including 122 primary IHCCs, 75 matched metastatic lymph nodes, and 122 adjacent non-cancerous liver tissues containing normal intrahepatic bile ducts (at least 5 cm distant from the tumor edge) were obtained from the Department of Pathology, Shandong Provincial Hospital. Immunohistochemical staining for CXCR4 was performed using the SABC kit (Boster, Wuhan, China) according to the manufacturer’s instruction. Primary antibody for CXCR4 (1:50, polyclonal, Abcam, MA, USA) was used for overnight incubation at 4°C. For the evaluation of CXCR4 IHC staining, a semi-quantitative scoring criterion was used, in which both the staining intensity and positive areas were recorded. A staining index (values 0–12), obtained as the intensity of CXCR4-positive staining (weak, 1; moderate, 2; strong, 3) and the proportion of immune-positive cells of interest (0%, 0; <10%, 1; 10–50%, 2; 51–80%, 3; >80%, 4), were calculated. The cases were grouped into low (score 0–6) and high (scores 8–12) CXCR4 expression. The study was approved by the Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong University, as stipulated by the Declaration of Helsinki, with written informed consent for the use of the specimens from all enrolled patients.

The sagittal sections containing the major downstream target regions of the rat prostriata were selected for c-fos IHC. These regions include the PrSd-PoS, LD, LP-Pul, PTN, and VLG and are directly innervated by the prostriata (Chen et al., 2021 (link)). In addition, we also evaluated c-fos expression in zona incerta (ZI) and substantia nigra (SN), which are not the targets of the prostriata. Selected sections were rinsed three times with 0.1 M PB and immersed in 0.3% hydrogen peroxide for 10 mins. Then the sections were rinsed three times again and blocked in 5% BSA for 60 mins. At the end, the sections were incubated with primary antibody against c-fos (200 μg/ml, 1:200, Boster Biological Technology, Wuhan, China) diluted with the 0.1 M PB overnight at 4°C. On the next day, the sections were incubated in the secondary antibodies (biotinylated goat anti-mouse/rabbit IgG, Boster) for 60 mins after thorough rinse with 0.1 M PB. The sections were then rinsed again and immersed in the Streptavidin-Biotin Complex solution (SABC kit, Boster Biological Technology) for 60 mins. After rinse with 0.1 M PB, the sections were incubated in 3, 3-diaminobenzidine (DAB) solution for 3 mins in a dark environment. Finally, the sections were mounted on the slides, dehydrated and coverslipped.

Immunohistochemistry (IHC) was performed using a ready‐to‐use SABC kit (Boster Biological Technology Co, Ltd). The sections were incubated with a murine monoclonal anti‐p65 antibody (Santa Cruz Biotechnology, Inc), a monoclonal rat anti‐F4/80 antibody (Santa Cruz) and a rabbit MCP‐1 antibody (Biosynthesis Biotechnology Co., Ltd), all for 48 hours at 4°C. The corresponding secondary antibodies (antimouse secondary antibodies for p65 antibody, anti‐rat secondary antibodies for F4/80 antibody, anti‐rabbit secondary antibodies for MCP‐1 antibody) were subsequently incubated for 1 hour at room temperature. Images were captured using a ZEISS Imager A1 Microscope (Carl Zeiss AG).

In brief, formalin-fixed, paraffin-embedded sections from 89 LSCC patients were deparaffinized and rehydrated. After antigen retrieval using citrate buffer (0.01 mmol/L, pH 6.0), the slides were washed three times with PBS and incubated in 10% normal goat serum to block nonspecific background staining. Sections were then incubated overnight with rabbit anti-human FOXA2 antibodies (Abcam, Cambridge, United Kingdom) at 4°C. After incubation, tissue sections were washed with PBS and treated with a streptavidin-biotin-peroxidase complex (SABC kit, Boster, Wuhan, China). Signal detection was performed using diaminobenzidine (DAB). Immunohistochemical staining was assessed semi-quantitatively by measuring intensity of the staining (0, 1, 2, or 3) and extent of staining (0, 0%; 1, 0–10%; 2, 10–50%; 3, 50–100%). Intensity scores and extent of staining were multiplied to give a weighted score for each case. For statistical analysis, weighted scores were grouped in two categories for which scores of 0–3 were considered negative and 4–9 were positive.

The procedure for BDA histochemistry was described in our previous study (Chen et al., 2020 (link)). Briefly, after rinses with 0.1 M of PB, the sections from the cases with BDA injections were incubated in 0.3% Triton X-100 in 0.1 M of PB for 60 min and in Streptavidin-Biotin Complex solution (SABC kit, Boster Biological Technology) for 120 min at room temperature in sequence. After rinses in 0.1 M of PB, the sections were visualized with 0.1 M of PB containing 0.05% DAB and 0.01% hydrogen peroxide. Then the sections were mounted on chrome alum and gelatin-coated slides, dehydrated in gradient alcohol and xylene, and finally coverslipped.

The procedure for BDA histochemistry has been described in our previous study (Lu et al., 2020 (link); Chen et al., 2021 (link)). In brief, the sections derived from the hemisphere with BDA injections were thoroughly rinsed (at least three times 10 min each) in 0.05 M PB. Then the sections were incubated in 0.3% Triton X-100 in 0.05 M PB for 1 h and in Streptavidin-Biotin Complex solution (SABC kit, Boster Biological Technology) for 3 h at room temperature in sequence. After rinse in 0.05 M PB for three times, the sections were visualized with 0.05 M PB containing 0.05% 3, 3-diaminobenzidine (DAB). Finally, the sections were mounted on chrome alum and gelatin-coated glass slides, dehydrated in gradient alcohol and xylene, and finally coverslipped.