Rnascope 2.0 hd detection kit red

Lens RNA transcripts were localized in situ using custom-synthesized oligonucleotide probes to Chmp4b (NM_029362.3, target region 136–1586 nt/bp Mm-Chmp4b Cat. # 418331) and the RNAscope 2.0 HD Detection Kit (RED) (P/N 310034, Advanced Cell Diagnostics, Inc, Hayward, CA) as described [Zhou and Shiels, 2018 (link)]. Briefly, mouse eyes were fixed (24 hr., 20°C) in neutral buffered formalin (10%, ThermoFisher) and paraffin-embedded. Protease pre-treated FFPE sections (5 μm) were hybridized (2 hr, 40°C) w ith target probe followed by signal amplification (15–30 min, 40°C) and chromogenic lab eling (15–30 min, 20°C) with alkaline phosphatase (AP)-conjugated Fast-Red label probe, than counterstained (Gill’s Hematoxylin-1) and imaged under a bright-field microscope (BX61, Olympus).

Lens RNA transcripts were localized using the RNAscope 2.0 HD Detection Kit (RED) (P/N 310034) and custom-synthesized oligonucleotide probes to Epha2 (NM_010139.3, target region 214–1758 bp) and Efna5 (NM_207654.2, target region 328–987 bp) according to the manufacturers’ instructions (Advanced Cell Diagnostics, Inc, Hayward, CA). Briefly, mouse eyes were fixed (24 hr, 20°C) in 10% neutral buffered formalin (Fisher Scientific) and processed using standard formalin-fixed-paraffin-embedded (FFPE) section techniques. Microtome sections (5 μm, RM2255, Leica Microsystems, Buffalo Grove, IL) on glass slides (SuperFrost Plus, ThermoFisher) were baked (1 hr, 60°C), de-waxed in xylene, dehydrated in ethanol, boiled in citrate buffer, then protease treated (10 μg/ml, 40°C, 30 min) in a HybEZ Oven (ACD). Pretreated sections were hybridized with target probes (2 hr, 40°C), followed by signal amplification oligonucleotides (15–30 min, 40°C). For chromogenic labeling, hybridized sections were treated with alkaline phosphatase (AP)-conjugated Fast-Red label probe (15–30 min, 20°C) and FastRed substrate (10 min, 20°C), then counterstained (Gill’s Hematoxylin-1/0.01% ammonia-H₂O), mounted (Acrymount, StatLab, McKinney, TX), and imaged under a bright-field microscope fitted with a digital camera (BX61, Olympus, Center Valley, PA).

Lung and liver samples were fixed in 4% formalin and paraffin embedded. Hematoxylin and eosin staining was performed using standard methods. Percent tumor area was calculated using ImageJ. ARNLT2 mRNA expression was assessed in human lung adenocarcinoma tissue microarrays (LC20813 and LC20815; US Biomax) with a custom RNAscope probe for ARNTL2 (Advanced Cell Diagnostics). RNA hybridization and development was performed according to manufacturer’s instructions using the RNAscope 2.0 HD Detection Kit (RED) using an Advanced Cell Diagnostics HybEZ™ Oven. ARNTL2 signal was scored as positive for red signal dots with high signal-to-background ratio, and crisp, clear edges. Only adenocarcinoma cores were included in our analysis. Cancer cell cytological differentiation was provided by the supplier.