After removal, tumours and lungs were fixed in 4% paraformaldehyde and then embedded in paraffin. 7 μm sections were deparaffinised with xylene and rehydrated with graded ethanol. The sections were stained according to routine immunohistochemistry procedures and visualised by Vectastain ABC or ABC-AP kit (Vector Laboratories, Burlingame, CA). Alternatively, samples were frozen in OCT and fixed in acetone/methanol before standard immunofluorescence procedures.
Primary antibodies used for immmunohistochemistry and immunofluorescence: Rat anti-CD31 at 1:200 dilution (BD Pharmingen; 550274), Rat anti-CD31 (DIANOVA; DIA-310), biotinylated mouse anti-SMA-alpha at 1:200 (Thermo Scientific; 14-9760-82), rat anti-NKp46 (BioLegend; 137606), rabbit anti-GLUT-1 (Abcam; ab652) at 1:500, rabbit anti-cleaved caspase-3 (Cell Signalling; 9661) at 1:500. The fluorochrome-conjugated Alexa 488 (A11070; A11006; A11017) and Alexa 568 (A11077; A11031) were used as secondary antibodies (1:200).
Lung metastases in the LLC model was analysed by Haematoxylin and Eosin (H&E) staining on 10 μm lung paraffin serial sections at day 14 post tumour injection26 (link).
Primary antibodies used for immmunohistochemistry and immunofluorescence: Rat anti-CD31 at 1:200 dilution (BD Pharmingen; 550274), Rat anti-CD31 (DIANOVA; DIA-310), biotinylated mouse anti-SMA-alpha at 1:200 (Thermo Scientific; 14-9760-82), rat anti-NKp46 (BioLegend; 137606), rabbit anti-GLUT-1 (Abcam; ab652) at 1:500, rabbit anti-cleaved caspase-3 (Cell Signalling; 9661) at 1:500. The fluorochrome-conjugated Alexa 488 (A11070; A11006; A11017) and Alexa 568 (A11077; A11031) were used as secondary antibodies (1:200).
Lung metastases in the LLC model was analysed by Haematoxylin and Eosin (H&E) staining on 10 μm lung paraffin serial sections at day 14 post tumour injection26 (link).
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