Vectra polaris pathology imaging system

Adipose tissues from patients or mouse tail tissues were isolated and fixed in 4% paraformaldehyde (DF0135, LEAGENE). The mouse tail tissues were decalcified in 10% neutral buffered EDTA (DD0002, LEAGENE) at 37 °C for 2 weeks. The adipose tissues and mouse tail tissues were dehydrated by ASP300S (Leica) and then were paraffin-embedded. The wax blocks were cut into 5-μm sections with RM2235 (Leica) for the subsequent staining. For all the staining experiments of mouse tail tissues, cross-sections of the mouse tails at almost the same level (located 1 cm distal to the surgical site) were used for both groups. The staining results were visualized on the Pannoramic SCAN (3DHISTECH) or the Vectra Polaris pathology imaging system (PerkinElmer).

Fresh tissue was immediately immersed in 10% neutral buffered formalin and was fixed at room temperature for 16–32 h. After rinsing with running water for 30 min, the tissue was dehydrated in gradient ethanol. After xylene transparent and paraffin embedment, tissue was sliced into 10-μm sections, and the slides were dried at room temperature overnight. Nucleotide probes of the target genes were designed and synthesized by Advanced Cell Diagnostics. smFISH was performed using a highly sensitive RNAscope^® technology based on the manufacturer’s instructions. The RNAscope^® Multiplex Fluorescent Reagent Kit v2(Cat. No. 323100) was applied for visualizing hybridization signals. Fluorescence images were captured on a confocal microscope (LSM780; Carl Zeiss, Germany) and the Vectra^® Polaris™ pathology imaging system (PerkinElmer, USA). The probes used for smFISH included: Hs-LYVE1 (Cat. No. 426911-C3), Hs-CD74 (Cat. No. 477521), Hs-IL1RN (Cat. No. 441601), Hs-ITGAM (Cat. No. 555091-C2), Hs-MYH11 (Cat. No. 444151), Hs-RGS5 (Cat. No. 533421-C4), Mm-Lyve1 (Cat. No. 428451), Mm-Cd74 (Cat. No. 437501-C2), Mm-Il1rn (Cat. No. 495101), Mm-Itgam (Cat. No. 311491-C3), Mm-Rgs5 (Cat. No. 430181), Mm-Myh11 (Cat. No. 316101-C2), negative control (Cat. No. 321831), and positive control (Cat. No. 321811).

We performed single‐molecule fluorescence in situ hybridisation (smFISH) on FFPE CM sections of 5 μm thickness using the RNAscope Multiplex Fluorescent Reagent Kit v2 (323100, Advanced Cell Diagnostics). Fluorescence signals were scanned with the Vectra Polaris pathology imaging system (PerkinElmer, USA). The target gene probes were as follows: Hs‐CDH5‐C2 (437451‐C2, Advanced Cell Diagnostics), Hs‐NPR3‐C3 (431241‐C3, Advanced Cell Diagnostics), Hs‐CCDC3‐C1 (1220531‐C1, Advanced Cell Diagnostics), Hs‐MIA (533581, Advanced Cell Diagnostics), Hs‐PDGFRA‐C3 (604481‐C3, Advanced Cell Diagnostics), Hs‐CPE‐C2 (454101‐C2, Advanced Cell Diagnostics) and Hs‐TSPAN8‐C2 (816551‐C2, Advanced Cell Diagnostics).