Dab reagent

Six animals in each group were anaesthetized with 2% pentobarbital sodium (40 mg/kg) and perfused with cold 0.9% NaCl solution followed by perfusate solution (4% paraformaldehyde, 2.5% glutaraldehyde, and 0.1 mol PBS). Brain stem contained locus coeruleus was cut out from brain tissues and fixed in 2.5% paraformaldehyde solution for 48 hours and was then cut into serial sections (5 μm). Preparation of tissue section and procedure of H&E staining were described in previous report [28 (link)].
For immunohistochemistry, the paraffin-embedded locus coeruleus sections were processed as free-floating slices, including deparaffinized, rehydrated, antigen retrieval, and then treated with hydrogen peroxidase in DDW (double distilled water) for 10 min at room temperature in order to inhibit endogenous peroxidase. Antigen retrieval was conducted by heating for 15 min. Slices were incubated with primary antibodies (anti-TH, 1 : 1000 dilution; anti-DBH, 1 : 1000 dilution; anti-CRF, 1 : 500 dilution) after blocking in the antisera. After incubation with the secondary antibody, sections were placed in DAB reagent (ZSGB-BIO, Beijing, China) for 5–10 min at room temperature. After a further rinsing in PBS, sections were restained with hematoxylin and were mounted on gelatin-coated slides for observation under a light microscope.

The formalin‐fixed, paraffin‐embedded tissues (n = 186) were cut into 5 μm sections. The sections were firstly stained with haematoxylin and eosin (H&E) using a standard protocol. For immunohistochemistry of Tn antigen, deparaffinized sections were boiled for 20 minutes in 0.01 mol/L citrate buffer at pH 6.0 for epitope retrieval. The sections were blocked using 0.3% H₂O₂ and 5% BSA. Then, sections were incubated overnight at 4°C with a specific anti‐Tn IgM monoclonal antibody (5 μg/mL, CA3638, clone 12A8‐C7‐F5, kindly provided by Dr. Tongzhong Ju, Emory University, Atlanta, Georgia, USA)13, 20, 26 followed by horseradish peroxidase‐conjugated antimouse IgM antibody (Abcam, ab97230) for 1 hour at room temperature. Finally, the sections were developed with DAB reagent (ZSGB‐BIO, China) and counterstained with haematoxylin.

The immunohistochemical methods have been described previously 19. Sections were deparaffinized, rehydrated, quenched of endogenous peroxidase, antigen retrieved and incubated with the indicated primary antibodies diluted by 1:100 overnight at 4°C. After extensively washing, slides were incubated with a secondary reagent, developed with the DAB reagent (ZSGB‐Bio Inc, Beijing, China), counterstained with haematoxylin and mounted. Negative controls were incubated without antibody or non‐specific rat IgG at an equivalent protein concentration. The histological specimens were examined by two senior pathologists blinded to the protocol. The score was determined according to the intensity of staining independently, as ranked from grade 0 to 3: 0, less than 10% of cells stained; 1, 10–50% of cells stained; 2, 50–75% of cells stained; 3, more than 75% of cells stained. For SDC1, only the membranes of surface epithelial cells and glandular epithelium cells with staining were scored. Meantime, tissue specimens were stained with haematoxylin and eosin and classified as normal mucosa, mild, moderate or severe enteritis (score 0–3), according to the proportion of sample crypt cross‐sections containing intraluminal or intra‐epithelial neutrophils.

The prepared paraffin‐embedded tissues were cut into 5 μm thick sections that were subsequently dewaxed and rehydrated. After antigen retrieval and blocking endogenous peroxidase, the sections were incubated with specific primary antibody against Tn antigen (2 μg/mL), E‐cadherin (1:500; Proteintech, 20874‐1‐AP) and Snail (1:500, Proteintech, 13099‐1‐AP) at 4°C overnight, followed by incubation with peroxidase labelled secondary antibody at 37°C for 30 minutes. The binding was visualized by the use of DAB reagent (ZSGB‐BIO, China) and the nuclei were counterstained with hematoxylin.

Brain sections were deparaffinized, hydrated, incubated with 3% hydrogen peroxide for 10 min, and then boiled in sodium citrate buffer for 5 min. Brain tissue sections were then blocked with 5% BSA at room temperature for 30 min and then incubated with anti-MAP2 (1: 400 dilution) antibody at 4°C overnight. Subsequently, the sections were treated with biotinylated anti-mouse secondary antibody (1: 500 dilution) at room temperature for 1 h, stained with DAB reagent (ZSGB-BIO, Beijing, China) for 5 min, and counterstained with hematoxylin for 2 min. Finally, the sections were analyzed by light microscopy (Nikon, Tokyo, Japan).

We collected normal cervical tissue and cervical cancer tissue at the Hunan Provincial Cancer Hospital for immunohistochemical staining, and it has been reviewed and approved by the Ethics Committee of Hunan Cancer Hospital. After dewaxing of sections, heat antigen retrieval was performed. The primary antibodies SFT2D1 (Immunoway, USA, 1:100) and CD31 (ZenBio, China, 1:100) were incubated overnight at 4 °C. The secondary antibodies were incubated for 20 min using the PV-9000 kit (ZSGB-BIO, China). DAB reagent (ZSGB-BIO, China) was used for antibody staining, with brown-yellow indicating positive signal areas. Cell nuclei were stained blue with hematoxylin (Servicebio, China). Images were captured using microscope (Zeiss, Germany) and analyzed using Image J software (1.53, USA).