RNA in situ hybridization probes targeting GIT1‐A (944‐985 bp; NM_001085454.1 ) and GIT1‐C (941‐983 bp; NM_014030.3 ) were designed by Advanced Cell Diagnostics (Hayward, CA, USA). Optimization of RISH probes and RISH probes targeting SRRM4 has previously been characterized.11 , 25 RISH results are available in Table S1 . RISH assays were carried out using the BaseScope assay kit (Advanced Cell Diagnostics) following the manufacturer's protocols. Red dots indicate positive RISH signals under 10× and 40× magnification. IHC assays on the CRPC TMA and scoring methods for RISH or IHC were carried out, as previously described.11 , 24 Briefly, RISH scores of 0, 1, and 2 indicate no positive signal, ≤20% positive signal, and >20% positive signal of cells within a tissue core, respectively. IHC scores were calculated by the signal intensity (no, low, medium, and high as 0‐3, respectively) multiplied by the percentage of positive cells—scores of ≥0.3 were considered to be positive. The SL801 autoloader and Leica SCN400 scanning system (Leica Microsystems; Concord, ON, Canada) were used to digitize the slides at a magnification of 40×. All antibodies used are listed in Table S2 .
Basescope assay kit
Manufactured by Advanced Cell Diagnostics
Sourced in United States
The BaseScope assay kit is a tool for detecting and visualizing RNA targets within cells or tissue samples. It utilizes a branched DNA technology to amplify and visualize target RNA molecules, enabling researchers to localize and quantify specific RNA transcripts with high specificity and sensitivity.
Automatically generated - may contain errors
2 protocols using basescope assay kit
1
RNA in situ Hybridization Probes for GIT1 Variants
2
RNA FISH Detection of BCRP3 in HeLa Cells
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RNA FISH was carried out using the BaseScope Assay kit (Advanced Cell Diagnostics, Newark, CA, USA) according to the manufacturer’s protocol. Briefly, HeLa cells cultured on slides were fixed with 4% paraformaldehyde at room temperature for 15 min, treated with hydrogen peroxide at room temperature for 10 min, and then digested with Protease III (1:15 dilution) at room temperature for 10 min. BCRP3-specific probes were custom produced by Advanced Cell Diagnostics (Cat#1055141-C1) and incubated with cells at 40℃ for 2 h in a HybEZ™ Oven (Advanced Cell Diagnostics), followed by multistep signal amplification as instructed in the protocol. Finally, cells were mounted and imaged using an Olympus FV3000 confocal microscope with 60x/1.40 oil objective lens.
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