Api deltavision deconvolution microscope

3D FISH was adapted from (Bonev et al., 2017 (link)). Briefly, HSPCs, T and EP cells were crosslinked with 1% PFA in PBS for 10 min at room temperature. After nuclei permeabilization, Chromosome FISH probes (Empiregenomics) were added to slide and sealed the coverslip with rubber cement. Cell DNA and probes were co-denaturated at 72°C for 2 minutes and hybridization was performed at 42°C overnight in Thermobrite (Abbot molecular). Cells were then washed 2 min at 72°C 0.3% Igepal/0.4XSSC and 1 min at RT 0.1%Igepal/2X SSC. After washing coverslips were mounted on slides with Vectashield (Clinisciences, France). Images were taken on an API Deltavision deconvolution microscope (Applied Precision).

Single molecule RNA fluorescence in situ hybridization (FISH) was performed using the QuantiGene ViewRNA ISH Cell Assay according to manufacturer’s instruction (Affymetrix). Images were taken on API Deltavision Deconvolution Microscope (Applied precision).