Fluorescent in situ hybridization with tyramide signal amplification (TSA) in cultured human cells

In brief, the DNA templates for making in situ RNA probes were first generated by RT-PCR using Trizol extracted total RNA from human 293 T cells as the template and gene-specific primers with T7 or T3 promoter sequence (sequences in Supplementary file 3). Digoxygenin-labeled antisense RNA probes were then generated by in vitro transcription and purified by ethanol precipitation (sequences in Supplementary file 1). Cells were fixed for ~18 hr with 70% ethanol at 4°C, rehydrated with 2x SSC (0.3 M NaCl, 30 mM trisodium citrate, pH 7.0) containing 65% formamide at room temperature, pre-hybridized for 1 hr with hybridization media (65% formamide, 5x SSC, 0.1% Tween-20, 50 µg/ml heparin, 500 µg/ml Type X tRNA, 9.2 mM citric acid) at 70°C, and hybridized for ~18 hr with hybridization media containing diluted antisense probes at 70°C. Cells were then successively washed with hybridization media, 2x SSC with 65% formamide, and 0.2x SSC at 70°C, and finally washed with 1x PBS at room temperature. For tyramide signal amplification, cells were washed with 1x PBS, incubated for 20 min with 100 mM glycine, pH 2.0, and washed with 1x PBS at room temperature. Cells were then incubated for 1 hr with blocking buffer (2% sheep serum, 2 mg/ml BSA, 0.1% Tween-20 in 1x PBS) at room temperature, and incubated ~18 hr with blocking buffer containing anti-digoxigenin-peroxidase antibody (1:500 dilution) at 4°C. Cells were washed successively with 1x PBS, borate buffer (100 mM boric acid, 37.5 mM NaCl, pH 8.5) with 0.1% Tween-20, and incubated for 5 min in tyramide reaction buffer (100 mM boric acid, 37.5 mM NaCl, 2% dextran sulfate, 0.1% Tween-20, 0.003% H₂O₂, 0.15 mg/ml 4-iodophenol) containing diluted Alexa Fluor tyramide at room temperature. Cells were washed successively with 1x quenching buffer (10 mM sodium ascorbate, 10 mM sodium azide, 5 mM Trolox in 1x PBS) and 1x PBS at room temperature. Coverslips were mounted using ProLong Antifade media (P7481, Life Technologies).