Sc 365900

Hypoxic microenvironment status, represented by CA-9, was compared between UFS and NAT groups to evaluate the alterations in physiological tumor conditions after NAT in pancreatic cancer.
Immunohistochemistry was performed on 10% formalin neutral buffer solution-fixed, paraffin-embedded tissue sections by Ventana autostainer model Discover XT (Ventana Medical System. A polyclonal goat antibody against human CA-9 antibody (1:300, sc-365900; Santa Cruz Biotechnology, Inc.) was used. In brief, tissue sections were incubated in citrate buffer for 4 min at 72°C to retrieve antigenicity, followed by incubation with the primary antibody. The bound primary antibody was incubated with the anti-goat secondary antibody at 37°C for 8 min and visualized using ultraView Universal DAB Detection Kit (760–500; Roche Diagnostics). The CA-9 sections were scanned using the NanoZoomer 2.0 system (Fig. 1F). The tumor regions without necrosis were encircled to evaluate the hypoxic tumor microenvironment status (Fig. 1G) and quantified using morphometric analysis from a color-detecting algorithm. CA-9 positivity was calculated as the percentage of CA-9-positive pixels out of the entire pixel count using morphometric analysis with the positive pixel count algorithm (Aperio ImageScope, version 12.4; Leica Biosystems) (Fig. 1H) (17 ).