D5d5r xp

FFPE sections (thickness of 4 μm) were deparaffinized in xylene and rehydrated in ethanol. Endogenous peroxidases were blocked with 0.3% hydrogen peroxide in methanol. Double staining was performed using the SignalStain IHC Dual Staining Kit (#36084, Cell Signaling Technology, Danvers, MA, USA). Slides were incubated with a primary mouse monoclonal anti-LAMP-3 antibody (clone 16H11.2, #3503452, Merck, Darmstadt, Germany; 1:500) at 4 °C overnight. Sections were then incubated at room temperature for 30 min with a secondary horseradish peroxidase (HRP)-conjugated mouse antibody (SignalStain Boost IHC Detection Reagent, Cell Signaling Technology). Visualization was performed with diaminobenzidine (SignalStain DAB Substrate Kit, Cell Signaling Technology). Antigens were retrieved by autoclaving for 10 min in Target Retrieval Solution (Agilent Technologies, Santa Clara, CA, USA) (100 °C, pH 9.0) for TIM-3. Slides were then incubated with a primary rabbit monoclonal anti-TIM-3 antibody (D5D5R XP, #45208, Cell Signaling Technology; 1:200) at 4 °C overnight. Slides were treated with SignalStain Boost IHC Detection Reagent (AP, Rabbit) (Cell Signaling Technology), and visualization was performed with Vibrant Red (SignalStain Vibrant Red Alkaline Phosphatase Substrate Kit, Cell Signaling Technology). Counterstaining was conducted with hematoxylin.

Four-μm thick FFPE sections were deparaffinized in xylene and rehydrated in ethanol. Endogenous peroxidases were blocked with 0.3% hydrogen peroxide in methanol. Antigens were retrieved by autoclave in 10 mM citrate buffer solution (105 °C, pH 6.0) for 5 min for VCAN, or by autoclave in Target Retrieval Solution (Agilent Technologies, Santa Clara, CA, USA) (100 °C, pH 9.0) for 10 min for TIM-3. Slides were incubated with primary rabbit polyclonal anti-VCAN antibody (HPA004726, Prestige Antibodies^® Powered by Atlas Antibodies, Sigma-Aldrich, St Louis, MO, USA; 1:500) or primary rabbit monoclonal anti-TIM-3 antibody (D5D5R XP^®; #45208; Cell Signaling Technology, Danvers, MA, USA; 1:400) at 4 °C overnight, and then detected using a horseradish peroxidase-coupled anti-rabbit polymer (K4003; Envision+ system, Agilent Technologies). Peroxidase was visualized with diaminobenzidine (Dojindo, Kumamoto, Japan), and nuclei were counterstained with hematoxylin.

The FFPE sections were deparaffinized and rehydrated. Antigens were retrieved by autoclave for 20 min in Target Retrieval Solution (Agilent Technologies) (100 °C, pH 9.0). Blocking was performed with 2% donkey serum in TBS/T for 30 min. Thereafter, the sections were stained with anti-human CD163 antibody (NCL-L-CD163; Leica biosystems, Wetzlar, Germany; 1:200) and anti-human TIM-3 antibody (D5D5R XP^®; #45208; Cell Signaling Technology; 1:200) at 4 °C overnight. The sections were incubated for 1 h with Alexa Fluor 488-conjugated anti-mouse (A-21202; Thermo Fisher Scientific, Waltham, MA, USA; 1:200) and Alexa Fluor 555-conjugated anti-rabbit (A-31572; Thermo Fisher Scientific; 1:200) secondary antibodies. Nuclei were stained with DAPI (D9542; Sigma-Aldrich; 40 ng/mL) for 10 min. Finally, the slides were mounted with ProLong Glass Antifade Mountant (P36984; Thermo Fisher Scientific), and images were obtained using an Olympus FV1000-D confocal microscope.