Aperio versa 8 tissue imaging system

Whole tissue sections from LC03 and LC07 (4‐μm‐thick formalin‐fixed, paraffin‐embedded) were stained with primary antibodies (EpCAM (#2929, CST, 1:500), CD163(#93498, CST, 1:200), Topoisomerase IIα (#12286, CST,1:400), TRIM29(17542‐1‐AP, Proteintech, 1:100), DKK1(Abcam, ab109416, 1:800) sequentially and paired with TSA 7‐colour kit (abs50015‐100T, Absinbio). The order of antibodies/fluorescent dyes was showed as following: anti‐ EpCAM/TSA 480, anti‐ CD163/TSA 780, anti‐TOPIIα/TSA 620, anti‐TRIM29/TSA 520, anti‐DKK1/TSA 570 and then by staining with DAPI (D1306; Thermo Fisher). Pictures were scanned with Aperio Versa 8 tissue imaging system (Leica). Images were analysed using Indica Halo software.

Multiplexed immunofluorescence (m-IF) was performed by sequentially staining frozen tissue sections with primary antibodies and paired with a TSA multiple-color kit (D110071-50T, WiSee Bio). For example, fixed frozen slides were incubated with anti-CD68 antibody (NB100-683, Novus, 1:100) for 30 min and then treated with anti-rat horseradish peroxidase-conjugated (HRP) secondary antibody for 10 min. IF labeling was developed for a strictly observed 10 min, using TSA 570 per manufacturer’s direction. Between all steps, the slides were washed with Tris buffer. The same process was repeated for antibodies/fluorescent dyes: anti-H3CIT (ab5103, Abcam, 1:500)/TSA 670, anti-CD68 (NB100-683, Novus, 1:100)/TSA 570. Each slide was treated with 2 drops of DAPI, washed in PBS, and manually coverslipped. Slides were air dried, mounted with an Anti-fade mounting medium, and taken pictures with Aperio Versa 8 tissue imaging system (Leica). Images were analyzed using Image J software.