Envision detection kit

Sections were treated with 3% (v/v) H₂O₂ in methanol to block endogenous peroxidase activity. Immunohistochemistry was performed using the Envision Detection™ kit (DAKO Agilent Technologies Inc., Santa Clara, CA, USA). Tissue sections were incubated with primary antibodies against IL-6, TNF-α, IL-1β, IL-17, phosphorylated (p) STAT3 (Tyr705), and TRAP for 2 h at room temperature. Sections were then incubated with a biotinylated secondary Ab and streptavidin–peroxidase complex for 30 min. The final colored products were developed using chromogen diaminobenzidine, and the sections were examined under a photomicroscope (Olympus, Tokyo, Japan). Two independent, blinded observers assessed the number of positive cells per section at high-power field (magnifications ×400). To analyze osteoclast parameters, osteoclast surface normalized to the bone surface were assessed by bone histomorphometric analyses (ImageJ software).

Immunohistochemistry was performed in 4 μm paraffin sections. The tissue sections were dewaxed in xylene, rehydrated in graded ethanol, and rinsed in distilled water. Citrate buffer (pH = 6.0) was used to retrive the antigen, and 0.3% H₂O₂ was used to block endogenous peroxidase activity. After the blockade of nonspecific binding, tissue sections were incubated with the Cyclophilin A antibody (cat. no. 5360s; 1 : 150 dilution; Santa Cruz Biotechnology, Inc.) and CD147 antibody (cat. no. 13287; 1 : 400 dilution; Cell Signaling Technology, Inc.) for overnight at 4 centigrade. The secondary antibody was applied using the Envision Detection kit (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA). The staining was performed by the application of diaminobenzidine tetrahydrochloride (DAB) for 2 min and hematoxylin for 1 min at room temperature. The stained sections were evaluated with a Nikon microscope in 10 independent fields at magnification, ×400. We followed the methods of Mu et al. [20 (link)].

Sections were treated with 3% (v/v) H₂O₂ in methanol to block endogenous peroxidase activity. Immunohistochemistry was performed using the Envision Detection™ kit (DAKO Agilent Technologies Inc., Santa Clara, CA, USA). Tissue sections were incubated with primary antibodies against IL-1β, TNF-α, and VEGF for 2 h at room temperature. Sections were then incubated with a biotinylated secondary antibody and streptavidin–peroxidase complex for 30 min. The final colored products were developed using chromogen diaminobenzidine. The sections were examined by light microscopy (Olympus, Tokyo, Japan). The number of positive cells was counted at high-power field (magnifications × 400) with the aid of Adobe Photoshop software by four individuals and averaged three randomly selected fields per tissue section.

We prepared TMA and performed IHC according to previously reported procedures^{47 (link)}. Immunostaining of TIM3 was performed using a mouse monoclonal anti-TIM3 antibody (Cat. No. 60355-1-lg, Proteintech Group, Wuhan, China) and the Envision detection kit (Dako, Carpinteria, CA, USA). All IHC staining was independently assessed by two experienced pathologists. The staining intensity was graded from 0 to 2 (0, no staining; 1, weak staining; 2 strong staining) (Fig. 1). The staining extent was graded from 0 to 4 based on the percentage of immunoreactive tumor cells (0%, 1–5%, 6–25%, 26–75%, 76–100%). A score ranging from 0 to 8 was calculated by multiplying the staining extent score with the staining intensity score, resulting in a negative (0–4) staining or a positive (6–8) staining for each example. To confirm the quality of the TIM3 antibody we used in this article, we also used this antibody to detect TIM3 expression and location in other tissues which were previously reported with a specific sub-cellular location. In addition, we also used TIM3 antibody from another commercial source (Cat. No. ab 185703, Abcam, USA) to validate the location of TIM3 in some metastatic prostate cancer tissues and normal prostate acinar cells.

Collagen 2 immunohistochemistry was performed as previously described58 (link) using mouse monoclonal anti-collagen 2 antibody (II-II6B3; DSHB Iowa) with Dako EnVision detection kit. In brief, slides were treated with 0.1% pronase (P-8811; Sigma) in PBS for 20 minutes at room temperature, followed by 2.5% hyaluronidase (H-3506, Sigma) in PBS for 30 minutes at 37 °C. Endogenous peroxidase activity was quenched with 3% H₂O₂ for 10 minutes at room temperature. Nonspecific binding sites were blocked with 10% goat serum in PBS for 60 minutes at room temperature. Sections were then incubated with primary antibody against collagen type 2 diluted 1:50 in PBS overnight at 4 °C. The slides were then washed in PBS, and incubated with the secondary antibody (Dako Real Envision/HRP, K5007) for 30 min at room temperature. DAB (Dako Real DAB+Chromogen, K5007) was applied for about 2 min and then removed by rinsing with distilled water. Slides were mounted with Biomount Aqua (Biognost, Croatia) and covered. Exclusion of primary antibody resulted in no staining.

Representative tumor tissues were sectioned and embedded in paraffin. The slides were then incubated with the primary antibody (mouse anti–8-oxoguanine monoclonal antibody, Abcam, Cambridge, UK) at 1:200 dilution overnight in a humidified chamber at 4°C. The slides were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Envision Detection Kit, Dako, Glostrup, Denmark) at 37°C for 30 minutes. Finally, the samples were stained with 3, 3-diaminobenzidine (DAB) solution and counterstained with hematoxylin and eosin (HE). Tumor cell death induced by capsaicin was detected by TUNEL assay with the In Situ Cell Death Detection Kit (Roche, Indianapolis, IN, USA) according to manufacturer’s instructions.