Rnascope target retrieval reagent

Antibody information, including dilutions and validations, are detailed in Supplementary Data 1. Tissue sections were deparaffinized and antigen retrieval was performed in 1X RNAscope Target Retrieval Reagent (Advanced Cell Diagnostics, Inc., Newark, CA, USA 322000) in a Decloaking chamber (Biocare Medical, Pacheco, CA, USA DV2004MX) for 15 min at 110 °C and 6 psi. IF staining was performed^{67 (link)}. For IHC, the Vectorstain Immunodetection kit (Vector Laboratories, Burlingame, CA, and HOH-3000) was used with modifications^{69 (link)–71 (link)}. In brief, following antigen retrieval, slides were treated in 0.3% hydrogen peroxide for 30 min to eliminate endogenous peroxidase activity. Non-specific antibody binding was inhibited by incubating sections with a serum-free protein-blocking solution. Tissue sections were incubated with primary antibody for 1 h at room temperature or overnight at 4 °C (p16 only). A secondary biotinylated multilink antibody was applied for 1 h, followed by streptavidin-peroxidase incubation for 30 min. Using freshly prepared 3.3’-diaminobenzidine (Vector Laboratories, Burlingame, CA, SK-4100) and DAKO Liquid DAB Substrate-Chromogen solution as a chromogen, the enzymatic reaction was visualized. Slides were counterstained with hematoxylin.

SARS-CoV-2-specific RNA in lung sections were determined by ISH assay as previously published.^{14 (link)} Briefly, 5 μm paraffin-embedded tissue sections were deparaffinized for 1 h at 60 °C. The endogenous peroxidases were quenched with hydrogen peroxide at room temperature for 10 min. Sections were boiled for 15 min in RNAscope Target Retrieval Reagents and incubated in RNAscope Protease Plus for 30 min before probe hybridization with RNAscope^® 2.5 HD Reagent Kit (Advanced Cell Diagnostics, USA). The tissues were counterstained with Gill’s hematoxylin and observed with a bright-field microscopy.

Sections (4 μm) of lung tumor tissue underwent deparaffinization, followed by treatment with RNAscope hydrogen peroxide for 10 min at RT, and 1× target retrieval reagent at 99°C for 15–30 min. The slides were then treated with RNAscope Protease Plus for 15–30 min at 40°C in the HybEZ Oven. After pretreatment, the slides were treated using the RNAscope 2.5 HD Detection Reagent-BROWN kit per the manufacturer’s protocol. After that, the signal was detected for either the negative control probe (dapB), or murine Cxcl9. The following reagents were used: RNAScope target retrieval reagents, Advanced Cell Diagnostics (ACD) #322000; RNAScope wash buffer reagents, ACD #310091; RNAScope 2.5 HD Detection Reagent-BROWN, ACD #322310; RNAScope H2O2 & Protease Plus Reagents, ACD #322330; RNAScope Negative Control Probe_dapB, ACD #310043; RNAScope Probe Mm-Cxcl9, ACD #489341; HybEZ II Oven, ACD #321710/321720; Humidity Control Tray, ACD #310012; EZ-Batch Wash Tray, ACD #310019; and EZ-Batch Slide Holder, ACD #310017. . Instrumentation used was as follows: Microscope/camera—Olympus BX41 System at 40×/0.65, ∞0.17/FN22; acquisition software—SPOT; and data analysis— FIJI.

Briefly, bake slides of patient tissues were obtained from The First Affiliated Hospital, Zhejiang University School of Medicine. The slides were pretreated using the RNAscope^® target retrieval reagents (322000), RNAscope^® H202 and protease reagents (PN 322381) (Advanced Cell Diagnostics, USA). Then, they were incubated with the probes targeting NAMPT (599311, Advanced Cell Diagnostics, USA), LYZ (421441, Advanced Cell Diagnostics, USA), positive control probe‐Hs (320861, Advanced Cell Diagnostics, USA), and negative control probe (320871, Advanced Cell Diagnostics, USA). The RNAscope^® multiplex fluorescent v2 detection kit (PN 323110) and RNAscope^® wash buffer (PN 310091) (Advanced Cell Diagnostics, USA) was used to detect target genes according to manufacturer recommendations.