Dab immunohistochemistry color development kit

Immunohistochemistry (IHC) was performed on paraffin-embedded human glioma and normal brain tissues collected from Shanghai Ninth People’s Hospital. The sections were deparaffinized in a xylene gradient and rehydrated in an ethanol gradient. Antigen retrieval was performed in sodium citrate buffer (10 mM sodium citrate pH 6.0) at 100 C for 20 min. Then, endogenous peroxidase was deactivated by applying 3% H₂O₂ in methanol. IHC of SYDE1 was performed by the Dako EnvisionTM method. Briefly, the sections (3 μm) were sequentially incubated with the anti-SYDE1 antibody (NBP1-89350, Novus Biologicals) and the HRP-conjugated secondary antibody (ab6721, Abcam). Then, the sections were color-developed with a DAB Immunohistochemistry Color Development Kit (E670033, Sangon Biotech) and counterstained with hematoxylin.
A Leica LF200 microscope was used to image the stained sections. The intensity of SYDE1 signals was scored as negative (0), weak (1) or strong (2). The staining extent of SYDE1 was evaluated according to the immunoreactive tumor cell percentage, which was scored as I (0%, score = 0), II (1–5%, score = 1), III (6–25%, score = 2), IV (26–75%, score = 3) and V (76–100%, score = 4). SYDE1 signals (ranging from 0 to 8) were calculated by multiplying the intensity score with the staining extent score and were classified into low (0–4) or high (5–8) groups for Fisher’s exact tests.

Excised tumor tissues were continuously sliced into 5-μm sections. The slides were dewaxed in xylene and dehydrated with a series of ethanol concentrations. Antigen retrieval was performed by treating the tissue sections with citrate buffer and heating them a microwave. The tissues were incubated with primary mouse monoclonal antibody against PCNA (dilution, 1:500; cat. no., 60097-1-Ig; Proteintech) overnight at 4°C. Next, they were incubated with HRP-labeled secondary antibody (Santa cruz, sc-516102) for 30 min and stained with the DAB immunohistochemistry color development kit (Sangon Biotech, China). Finally, the dyed tissue sections were examined under a microscope (Olympus, Japan).

The cells were inoculated in 100 mm culture dishes and incubated for 48 h after drug treatment. After sufficient lysis of the cells, the supernatant was collected by centrifugation. The protein content was determined using the BCA kit (Sigma-Aldrich). SDS-PAGE electrophoresis was performed to separate proteins with different relative molecular masses, which were then transferred onto PVDF membranes (Thermo Fisher Scientific). The proteins were blocked with 5% skim milk for 2 h, washed 3 times with TBST, and incubated with primary antibodies (GPX4, AR, FKBP51, PSA, LK2, S100P, TMPRSS2, Bax, caspase-3, Bcl-2, GPX1, AR, PSA, 1:1000, all purchased from Abcam) overnight at 4°C. PVDF membranes were washed with TBST, and peroxidase-labeled secondary antibody (1:10000, Abcam) was added, incubated for 2 h at room temperature, and washed 4 times with PBST. The proteins were visualized by a DAB immunohistochemistry color development kit (Sangon Biotech, China) and photographed using a gel imaging analysis system (Thermo Fisher Scientifi). Relative protein levels were calculated by ImageJ as previous report (Sun et al., 2022 (link)).

The xenograft tumors were embedded in paraffin and cut into 4-μm thick sections. The sections were then dewaxed with xylene, hydrated with gradient ethanol, and incubated with primary anti-Ki67 antibody (Bioss Antibodies, Inc., 1:200) at 4 °C overnight. On the next day, the sections were incubated with secondary antibody at room temperature for 1 h. The expression of Ki-67 was detected using a DAB immunohistochemistry color development kit (Sangon Biotech, China) following the manufacturer’s instructions. Cell images were captured under a fluorescence microscope (IX-51; Olympus) at 200x magnification.