Envision detection kit

Slides (3 slides per sample) from formalin-fixed paraffin-embedded lung adenocarcinoma tissues were used for the histologic evaluation of PKM2 and PD-L1 expression by IHC. Briefly, 4-μm tissue sections were deparaffinized and subjected to heat-induced antigen retrieval. The slides were incubated overnight at 4 °C with a rabbit anti-human PKM2 mAb (1:100; D78A4, Cell Signaling Technology, Danvers, MA, USA) or rabbit anti-human PD-L1 mAb (1:100; E1L3N, Cell Signaling Technology). After incubation with horseradish peroxidase-conjugated secondary antibody (Envision™ Detection Kit, GK500705, Genetech), diaminobenzidine was used to visualize the staining.

On days 0, 1, 3, 7, and 13 after therapy initiation, in order to minish the sampling error of IHC staining, three U87MG tumor-bearing mice in each group were sacrificed and tumor samples were collected to fix in 10% formalin neutral buffer solution for paraffin embedding. Paraffin-embedded tissues were cut into 4 μm sections and stained with mouse anti-human Ki67 antibody (1 : 100, Abcam) and rat anti-mouse anti-CD31 (Abcam). Endogenous peroxidase activity was blocked with 3% H₂O₂ for 15 min. Antigen retrieval was performed by boiling the sections for 10 min in citrate buffer (pH 6.0) and cooling at room temperature, followed by blocking with 10% normal goat serum for 1.0 h. The sections were incubated with optimal dilutions of anti-Ki67 and anti-CD31 overnight at 4°C, and then Ki67⁺ cells and CD31⁺ areas were detected with horseradish peroxidase- (HRP-) conjugated anti-mouse/rat secondary antibodies using an EnViSion Detection kit (Gene Tech Co., Ltd., Shanghai, China). After washing with PBS three times for 5.0 min each time, the immune complexes were visualized using a Peroxidase Substrate DAB kit (Gene Tech Co., Ltd., Shanghai, China) according to the manufacturer's instructions. Finally, the slices were counterstained with hematoxylin and dehydrated. Ki67⁺ cells and CD31⁺ areas were counted on 4 randomly selected visual fields per section of each sample under high power.

Serial paraffin-embedded sections (2 μm thick) from the 158 patients were de-waxed with xylene and subsequently hydrated with an ethanol gradient. For antigen retrieval, the tissue sections were immersed in EDTA (1 mmol/l, pH 9.0) and maintained at 100°C for 15 minutes, before cooling at room temperature for 2 h. The sections were then washed with phosphate-buffered saline (PBS, pH 7.4) and immersed in 3% H₂O₂ for 15 min to eliminate endogenous peroxidase activity. After incubation in 10% normal goat serum (Invitrogen, Paisley, UK) for 30 min at room temperature to block non-specific antigens, sections were then incubated overnight at 4°C with the primary detection antibody (monoclonal mouse anti-human CTLA-4, Abcam, US, ab134090, 1:500 dilution). Excess antibody was removed by gentle rinsing, and the sections were washed with PBS three times. Subsequently, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (EnVision™ Detection Kit, GK500705, Gene Tech) at room temperature for 30 min. After washing three times with PBS, sections were stained with 3,3′-diaminobenzidine (DAB) for 1 min, and nuclei were counterstained with hematoxylin. Slides were dehydrated in an ethanol gradient, mounted with neutral gum and stored at room temperature for later observation.

Ovarian normal and cancer tissues were obtained from the tissue bank of Fudan University Shanghai Cancer Center (FUSCC). This study was approved by the Clinical Research Ethics Committee of Fudan University Shanghai Cancer Center and conducted with an informed consent signed by each participants before the use of tissues (No. 1711178-23) according to the institutional guidelines. For immunohistochemistry (IHC), human or xenograft tumor samples were fixed in 10% formalin and embedded in paraffin wax. Unstained 3-mm sections were then cut from the paraffin blocks for IHC analysis. The sections were stained overnight at 4 °C with anti-ID1 (1:200), anti-LC3B (1:200), and anti-ATF6 (1:200). The secondary antibodies against mouse or rabbit IgG were obtained from an IHC kit, EnVision™ Detection Kit (GENE, USA). Diaminobenzidine (DAB) was used for coloration, and the color with dark brown was considered to be the strong positive staining.

Xenograft tumor samples were fixed in 10% formalin and embedded in paraffin wax. 4-mm sections were cut from the paraffin blocks. The sections were then stained for 6 h at 4 °C with anti-ECM1 (1:150) or anti-p65 (1:150). The secondary antibody against mouse/rabbit IgG was from an EnVision™ Detection Kit (GENE, San Francisco, CA, USA). For coloration, the dark brown color by Diaminobenzidine (DAB) represents a strong positive staining.

Immunohistochemical staining (IHC) were performed on formalin-fixed, paraffin-embedded tissue sections obtained from HCC patients according to standard procedures. The sections were blocked for 30 min using 10% normal goat serum, and they were separately incubated with the primary antibodies directed against SAA1 at 37°C for 3 hours. After washing, sections were incubated for 30 min with biotinylated secondary antibody (Envision™ Detection Kit; Gene Tech, Shanghai, China) at 37°C. The staining of sections was performed using the streptavidin-biotin-peroxidase complex for SAA1. The complex was visualized with diaminobenzidine (DAB) and counterstained with hematoxylin. The sections were then dehydrated in a graded series of alcohol, cleared in xylene, and mounted onto glass slides. The staining was quantified by digital image analysis with Image-Pro Plus 6.0 software (Media Cybernetics, MD, USA).