Real envision detection kit

For histologic analysis, lung sections were stained with hematoxylin and eosin and photographed using a light microscope (Olympus). Immunostaining of GABARAPL1 and ERβ of tumor sections was performed using a Real Envision Detection kit (GeneTech, Shanghai) according to the manufacturer’ s instruction. Data analysis was performed blindly.

Lung specimens fixed in 10% buffered formalin and were embedded in paraffin blocks. Paraffin-embedded lung sections were heat-fixed, deparaffinized, rehydrated, antigen retrieval, blocked with 3% goat serum and incubated with anti-α-SMA antibody (1:200, Cell Signaling Technology, MA) or anti-COL1A1 antibody (1:200, Cell Signaling Technology, MA) or anti-E-cadherin antibody (1:150, Cell Signaling Technology, MA) or anti-N-cadherin antibody (1:150, Cell Signaling Technology, MA) overnight at 4 °C, then the slides were detected using Real Envision Detection kit (GeneTech, Shanghai, China) according to the manufacturer’s instructions. Observe the sections with a microscope and take pictures. Image J software calculated the ratio of positive expression area to the total field of immunohistochemical staining of α-SMA, COL1A1, E-cadherin and N-cadherin.

Biopsy samples were fixed in 10 % formalin and paraffin-embedded. Then, the samples were cut into 5-μm-thick sections. Immunohistochemistry was performed according to the appropriate protocols as previously described [14 (link)]. The following antibodies were used as primary antibodies: anti-human CD66b (Anaspec, CA, USA), anti-human CD3 (Gene Tech, Shanghai, China), anti-human PD-L1 and anti-human PD-1 (BD Bioscience, San Jose, USA). A mouse anti-human CD66b monoclonal antibody was used to identify neutrophils (1:50), and rabbit anti-human CD3 monoclonal antibody was used to identify T cells (1:100). A Real Envision Detection Kit (Gene Tech, Shanghai, China) was used for detection. Quantification of immune-cell infiltration was then determined.

The tissue microarray of human breast cancer (BR811) was purchased from US Biomax, Inc (Rockville, MD). A tissue microarray containing 80 paired human breast cancer tissues (Cat No. BRC1602) was purchased from the shanghai Superbiotek Pharmaceutical Technology Co., Ltd. (Shanghai). Paraffin-embedded sections were stained with the antibody against CYTL1 and then determined using the Real Envision Detection kit (GeneTech, Shanghai) according to the manufacturer’s instructions. Data analysis was performed blindly.

Xenograft tumor and matrigel plug sections were deparaffinized with xylene and rehydrated with addition of ethanol. Heat-induced antigen retrieval was performed in Tris-EDTA buffer. Tissue sections were subjected to 3% hydrogen peroxide to remove endogenous peroxidase activity and then blocked with blocking buffer (P0102; Beyotime, China). Slides were incubated with primary antibodies against CD31 (sc-1506; Santa Cruz Biotechnology, Dallas, Texas, USA), VWF (sc-14014; Santa Cruz Biotechnology), CD34 (#3569; Cell Signaling Technology, MA, USA), and GFP (ARH2068; Antibody Revolution, San Diego, CA, USA) at 4°C overnight. Sections were washed three times for 5 minutes each time. For immunofluorescence analysis, fluorescein-labeled secondary antibodies (20014; 20106; Biotium, Hayward, CA, USA) were incubated at room temperature for 1 hour. Before immunofluorescence analysis with a confocal microscope, cells were counterstained with DAPI. For immunohistochemistry analysis, Real Envision Detection Kit (GK500710; Gene Tech, Shanghai, China) was used and signals were visualized through the diaminobenzidine reaction.

Immunostaining of CD31 was performed using a Real Envision Detection kit from the Gene Tech Company according to the manufacturer's instructions. H&E staining in tumor tissues was performed following the manufacturer's protocol. A TUNEL assay was performed to detect apoptotic cells using the TUNEL BrightGreen Apoptosis Detection kit from Vazyme (Piscataway, NJ) according to the manufacturer's instructions.