Quantigene viewrna ish cell assay kit

To quantify mRNA levels via FISH the QuantiGene ViewRNA ISH Cell Assay kit (Affymetrix, USA) was used, essentially following the manufacturer’s instructions. At least 1x 10⁷ trypanosomes were harvested, fixed with 4% (w/v) formaldehyde (FA) for 10 minutes at room temperature and, subsequently, washed two times with PBS. Cells were allowed to settle on poly-l-lysine-coated slides (within hydrophobic circles) for 30 minutes. For protease digestion the settled cells were incubated with the protease solution (1:1 600 in PBS) for 15 minutes at 25°C. The following probes for mRNA detection were used in a 1:100 dilution of the original stock: eGFP (full antisense ORF, red = type 1) and ESAG6 (antisense to nucleotides 107–1206 Tb427.BES40.3, red = type 1). Only samples from the same slide were compared for quantification of mRNA levels. Per slide, fixed cells (non-induced slender, density-induced stumpy and ectopic VSG overexpression induced for 24 and 48 hours) of a proliferating or growth arrested clone were incubated either with the ESAG6 or eGFP probe.

Cells were grown on a 15 mm, poly-L-lysine-coated glass coverslip. At ~70% confluence, cells were serum starved in 8% charcoal-stripped media for 48 h, followed by 48 h treatment with 10 nM E2. At the end of treatment, cells were fixed in 4% formaldehyde, dehydrated by an ethanol gradient (50–100%) and stored at −20 °C. For the hybridization assay, cells were rehydrated by an ethanol gradient (100–50%) into PBS. Between subsequent steps, cells were washed with PBS. The Affymetrix QuantiGene ViewRNA ISH cell assay kit was used for NEAT1 staining. Cells were permeabilized by 5 min incubation at RT in Detergent Solution QC and digested for 10 min at RT by Protease QS (1:4,000 in PBS). Next, the target-specific Probe Set (1:100 in Diluent QF) was allowed to hybridize for 3 h at 40±1 °C. Between subsequent steps, cells were washed by soaking in Wash Buffer. Sequential hybridization steps were conducted for signal amplification—PreAmplifier Mix (1:25 in Diluent QF), Amplifier Mix (1:25 in Diluent QF) and Label Probe Mix (1:25 in Diluent QF), each incubated 30 min at 40±1 °C. After two 10-min washes in Wash Buffer, nuclei were stained with 4',6-diamidino-2-phenylindole and cover slips were mounted to slides with Prolong Gold Antifade Reagent (Life Technologies) for visualization.