Immunohistochemistry kit

Tissues were sent to make paraffin section by the Nanjing Drug Toxicology Research Institute. Blocking was performed by incubating 2 h at room temperature with PBS containing 10% goat serum. Then, sections were incubated with primary antibodies for 2 h at 4 × degrees celsius. Then the immunohistochemistry kit of Maixin Biotech was used. The immunoreactivity was finally examined by NanoZoomer 2.0 RS (Hamamatsu Photonics Co., Ltd, Shizuoka, Japan).

Immunohistochemical staining was conducted using Immunohistochemistry Kit (Maixin, China). Briefly, 4 μm-thick histology sections were deparaffinized and hydrated. Antigen retrieval was performed by boiling at 100 °C for 10min in 10 mmol/l citrate buffer (pH = 6.0). Then sections were incubated with polyclonal antibody against human ATG12 (Abcam, Cambridge, UK), LC3 (Abcam, Cambridge, UK) overnight at 4 °C. Sections were then incubated with horseradish-peroxidase-conjugated anti-goat secondary antibody (DakoCytomation, Glostrup, Denmark) for 1 h at room temperature. The visualization signal was developed with 3,3-diaminobenzidine tetra hydrochloride staining, and the slides were counterstained in hematoxylin and observed under a light microscope (Olympus, Japan). For staining of ATG12 and LC3, the scores of positively labeled cell were recorded as: 0 (no staining, 0%), 1 (staining range, 1–25%), 2 (staining range, 26–50%), 3 (staining range, 51–75%) or 4 (staining range, 76–100%). Intensity scores were recorded as: 0, no labeling; 1, weak labeling; 2, moderate labeling; or 3, strong labeling. The multiplication of the above two scores representing the results as follows: 0–4 represents low expression, 5–12 represents high expression.

The paraffin sections were deparaffinized, followed by hydration, using an immunohistochemistry kit (Maixin Biotech. Co., Fuzhou, China) according to the manufacturer instructions. LIMK1, destrin, E-cadherin, vimentin, Ki-67, and CD34 antibodies (Abcam, Cambridge, UK) were incubated overnight at 4 °C, and normal rabbit immunoglobulin G was considered as a negative control. The specimens were visualized with 3,3′-diaminobenzidine and counterstained with hematoxylin. The staining was brown or tan and located in the cytoplasm or nucleus. The scores according to the degree of positive staining and the percentage of stained cells were as follows: 0, no coloring; 1, light brown; 2, dark brown; 0, staining cells < 5%; 1, staining cells between 5% and 25%; 2, staining cells between 26% and 50%; 3, staining cells > 50%. The score was obtained by adding the intensity and reactivity. The scores < 2 represented low expression, the scores ≥ 2 represented the high expression.

Following deparaffinization steps, including baking, xylene treatment, and a gradient alcohol series, antigen retrieval was conducted on tissue chip specimens (HStmA160CS01, OUTDO Biotech, Shanghai, China) using citrate buffer. Subsequently, the samples underwent the following procedures: removal of hydrogen peroxidase with a 3% hydrogen peroxide solution for 10 min, followed by blocking with goat serum for 10 min. The ANXA5 primary antibody (Proteintech, Wuhan, China) was then incubated overnight at 4 °C, and immunohistochemistry staining was carried out utilizing an immunohistochemistry kit from Maixin Biotech (Fuzhou, China).
The staining degree was assessed by categorizing the percentage of stained area into four grades: Grade 1 (≤ 25%), Grade 2 (25–50%), Grade 3 (50–75%), and Grade 4 (≥ 75%). Simultaneously, staining intensity was classified into four grades: Grade 0 (no staining), Grade 1 (weak positivity), Grade 2 (moderate positivity), and Grade 3 (strong positivity). These two scores were then multiplied together to generate a weighted score, which ranged from 0 to 12. The results were independently evaluated by two pathologists based on both staining intensity and degree.