Dab substrate system

Four micrometer-thick slides were deparaffinized with xylene before rehydration using an ethanol gradient. Quenching of the endogenous peroxidase activity was achieved by incubating samples with endogenous peroxidase blocker. After blocking with 3% BSA, sections were incubated with mouse anti-α-SMA (Cell Signaling Technology, Boston, MA, USA), mouse anti-TGF-β (R&D, Minneapolis, MN, USA), rabbit anti-VEGFA (Abcam, Cambridge, UK), and mouse anti-FGF basic/FGF2 (Novus, Littleton, CO, USA) overnight at 4 °C. Horseradish peroxidase-conjugated goat anti-mouse/rabbit lgG was used as the secondary antibody. The DAB Substrate System (DAKO) was used to reveal the immunohistochemistry staining.

Paraffin-embedded testis tissue sections were soaked in xylene for 10 min two times. Then slides were dehydrated with an alcohol gradient concentration (100%, 95%, 90%, 70%, 50%, and 30% alcohol for 3 min per wash). The slides were incubated with PBS containing 3% H₂O₂ for 10 min to quench endogenous peroxidase activity. Triton (0.3%) was added to the glass slide for drilling for 10 min. After doing that, the slides were sealed at 37° C for 1 h in PBS containing 3% BSA and incubated with rabbit anti-LHR (Affinity, USA) at 4° C overnight. Whereafter, the slides were incubated in HRP-conjugated secondary antibodies (Boster, Wuhan, China) at 37° C for 1 h. Finally, we used the DAB Substrate System (DAKO) to observe the immunohistochemical staining under a DXM12000F microscope (Nikon, Tokyo, Japan).

Five-μm-thick paraffin-embedded sections were deparaffinized with xylene (twice for 5 minutes) before being rehydrated in water using an ethanol gradient. After washing with water, antigen retrieval was performed in a steamer using citrate buffer (pH 6.0; DAKO) for 20 minutes, and the samples were then cooled to room temperature. The sections were then washed with TBST buffer and incubated with 3% H₂O₂ for 10 minutes and blocked with avidin/biotin blocker and serum-free blocking reagent. The sections were subsequently incubated with rabbit anti-α-SMA antibody (Abcam) overnight at 4 °C. The DAB substrate system (DAKO) was used to detect the immunohistochemical staining. 3DHISTECH pannoramic viewer was used for viewing digital slides.

MCT1, MCT4, CAIX and Ki67 protein expression for U251 shMCT1 and U251 shCTRL tumours collected from NSG mice were evaluated by immunohistochemistry (IHC). IHC for MCT1 was performed using UltraVision LP detection system HRP Polymer (Thermo Fisher Scientific, Waltham, MA, USA), and for MCT4, CAIX and Ki67 using UltraVision Large Volume Detection System Anti-Polyvalent, HRP (Thermo Fisher Scientific), as previously described [21 (link),23 (link)]. Briefly, deparaffinised and rehydrated slides were submitted to heat-induced antigen retrieval for 20 min at 98 °C with 10 mM citrate buffer (pH 6.0). After endogenous peroxidase inactivation, incubation with the primary antibody was performed overnight for MCT1, and during 2 h for MCT4, CAIX and Ki67, at room temperature. The immune reactions were visualized with 3,3′-diamonobenzidine (DAB + Substrate System; Dako, Denmark) as a chromogen. The slides were counterstained with haematoxylin and mounted with Entellan^® (Merck-Millipore, Darmstadt, Germany). For each immunoreaction, a positive control was included.

Immunohistochemistry was performed as previously described [43 (link)]. The primary antibodies used were as follows: rabbit anti-V-ATPase (1:100, ab157458 Abcam) and rabbit anti-CAXII (1:200, ab195233, Abcam). IHC was performed with the Ultravision detection system anti-polyvalent, HRP (Lab Vision Corporation). After deparaffinization and rehydration, the slides were heated for 20 min at 98 °C with 10 mM citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidase was inactivated, and the tissues incubated with the primary antibody for 2 h at room temperature. The immune reaction was visualized using the chromogen 3,3′-diaminobenzidine (DAB+ substrate system; Dako). Tissue sections were counterstained with Gill-2 hematoxylin. For negative controls, primary antibodies were replaced by a universal negative control antibody (N1699, Dako). Positive controls were normal colon tissue for CAXII and breast cancer tissue for V-ATPase. Immunoreaction was evaluated using a semiquantitative score system, considering the extension and intensity of staining, as previously described [43 (link)]. Some of the cases could not be evaluated for immunoreaction, due to missing TMA samples or to insufficient representation of the tumor.

Five μm-thick slides were deparaffinized with xylene before rehydration using an ethanol gradient. Next, quenching of endogenous peroxidase activity was achieved by incubation with 3% H₂O₂. After blocking with 3% BSA, the sections were incubated with mouse anti-α-SMA overnight at 4 °C. The secondary antibodies incubated were horseradish peroxidase-conjugated goat anti-mouse IgG (Boster). The DAB Substrate System (DAKO) was used to reveal the immunohistochemical staining.
TUNEL assay as described by the manufacturer (Roche Applied Science, Mannheim, Germany), and the nuclei were stained with 4′ 6-diamidino-2-phenylindole (DAPI) (Sigma). The images were captured using a laser scanning confocal fluorescence microscope (Olympus).