Carbonic anhydrase 9

H&E, picrosirus red, IHC, and IF staining were performed using standard techniques on formalin-fixed paraffin-embedded sections. Tissues for quantitative evaluation were processed in parallel. For quantification, whole tissue sections were imaged on a Keyence BD microscope with an automated stage. The whole virtual slide was used for quantification using the ImageJ software package (rsbweb.nih.gov/ij/).
Quantification of PSR staining was performed using ImageJ. RGB (Red,Green,Blue) images were split in the three-color channels. The green channel was used for quantification of the relative area that displayed a signal above a certain, constant threshold.
Antibodies used for IHC/IF or WB: cleaved caspase-3 (Cell Signaling Technology Cat# 9661, RRID:AB_2341188), Carbonic Anhydrase IX (Santa Cruz Biotechnology Cat# sc-25599, RRID:AB_2066539)), Hif1α (Novus Cat# NB100–131H, RRID:AB_1108863), CD31 (Santa Cruz Biotechnology Cat# sc-28188, RRID:AB_2267979), CD34 (Abcam Cat# ab8158, RRID:AB_306316), Collagen IV (Bio-Rad / AbD Serotec Cat# 2150-1470, RRID:AB_2082660), Ki-67 (Abcam Cat# ab16667 RRID:AB_302459), LOX (IMGENEX Cat# IMG-6442A RRID:AB_1930256), LOXL2 (Biorbyt Cat# orb41134 RRID:AB_10987961), β-Actin (Santa Cruz Biotechnology Cat# sc-1615 RRID:AB_630835).

H&E, IHC and IF staining was performed using standard techniques on formalin fixed paraffin embedded sections. Tissues for quantitative evaluation were processed in parallel. For quantification whole tissue sections were imaged on a Keyence BD 6000 microscope with an automated stage using a Nikon 10 × objective. The individual images were stitched using the Keyence Analyzer software, to obtain a virtual slide. The whole virtual slide was used for quantification using the ImageJ software package (rsbweb.nih.gov/ij/).
Immunofluorescence images were acquired on a Nikon A1 laser-scanning confocal microscope using a Nikon 20 × objective. Image processing and quantification was performed using the ImageJ software package (rsbweb.nih.gov/ij/).
Antibodies used for IHC, IF or WB: Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID:AB_2341188), Carbonic Anhydrase IX (Santa Cruz Biotechnology Cat# sc-25599, RRID:AB_2066539)), CD31 (Santa Cruz Biotechnology Cat# sc-28188, RRID:AB_2267979), CD34 (Abcam Cat# ab8158, RRID:AB_306316), Ki67 (Abcam Cat# ab16667 RRID:AB_302459).