Tumours were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. For H&E staining, 5-μm-thick sections were cut and were examined by light microscopy (10 × and 40 × magnification).
Using a microtome, 4-µm-thin tissue slices were cut from tumours and transferred to gelatine-coated slides. The paraffin‐embedded tumour tissue sections were incubated overnight at 4°C with anti‐STAT3 (Santa Cruz Biotechnology, USA), anti‐CD163 (Proteintech, USA), anti‐TGF‐β (Santa Cruz Biotechnology, USA), anti‐vimentin (Invitrogen, USA), anti‐IL‐10 (Invitrogen, USA), and anti-CXCL12 (Novusbio, USA) (all in a dilution to 1:300). Slices were rinsed in PBS and treated with a secondary antibody conjugated to streptavidin/Haptoglobin-Related Protein (HRP) (Biocare Medical, Concord, CA, USA). Immunoreactivity to various proteins was visualized using a colorimetric-based detection kit (TrekAvidin-HRP Label + Kit from Biocare Medical, Pacheco, USA) according to the manufacturer's procedure. The digital images were captured using a high-power objective (40 ×) and a light microscope (Nikon Eclipse 2000 equipped with Nikon DS-Fi2; Nikon Corporation, Tokyo, Japan). The following scale was used to as-sess the intensity of cell immunostaining: Absence of positive cells (#1), small number of positive cells (#2), moderate number of positive cells (#3), and substantial number of positive cells (#4). Two previously trained examiners performed a double-blind evaluation of labelling intensity [18] (link).
Using a microtome, 4-µm-thin tissue slices were cut from tumours and transferred to gelatine-coated slides. The paraffin‐embedded tumour tissue sections were incubated overnight at 4°C with anti‐STAT3 (Santa Cruz Biotechnology, USA), anti‐CD163 (Proteintech, USA), anti‐TGF‐β (Santa Cruz Biotechnology, USA), anti‐vimentin (Invitrogen, USA), anti‐IL‐10 (Invitrogen, USA), and anti-CXCL12 (Novusbio, USA) (all in a dilution to 1:300). Slices were rinsed in PBS and treated with a secondary antibody conjugated to streptavidin/Haptoglobin-Related Protein (HRP) (Biocare Medical, Concord, CA, USA). Immunoreactivity to various proteins was visualized using a colorimetric-based detection kit (TrekAvidin-HRP Label + Kit from Biocare Medical, Pacheco, USA) according to the manufacturer's procedure. The digital images were captured using a high-power objective (40 ×) and a light microscope (Nikon Eclipse 2000 equipped with Nikon DS-Fi2; Nikon Corporation, Tokyo, Japan). The following scale was used to as-sess the intensity of cell immunostaining: Absence of positive cells (#1), small number of positive cells (#2), moderate number of positive cells (#3), and substantial number of positive cells (#4). Two previously trained examiners performed a double-blind evaluation of labelling intensity [18] (link).
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