Brown Nat Protocols Fig 6

Abstract

Figure 6 | RASER-FISH hybridization examples with inset magnifications. a, Deconvolved widefield image of plasmids pCx (green) and pEx (red) from the α-globin gene region36 hybridized to C127 nucleus detected with DAPI. b, Fosmids recognising NKX2 (green) and Pax2 (red) hybridized to mouse ES cells39 with nuclei detected with DAPI. c, BAC RP24-217l105 (red) hybridized to C127 nucleus with chromatin stained with SYTOX Green (grey), imaged by 3D-SIM. Orthogonal (top) and lateral (bottom) cross sections are shown. d, 3D-SIM image of a C127 nucleus hybridized with 6 pools of oligonucleotide probes directly labelled with Atto 565, Abberior Star Red and Oregon Green and covering 1030 kb of the α-globin gene region37. Maximum projection with the nuclear boundary defined by DAPI outlined (DAPI not shown). e, 3D-STED image (maximum intensity projection) of a mouse erythroblast nucleus hybridized with 3 pools of oligonucleotide probes directly labelled with Atto 565, Abberior Star Red and Oregon Green and covering 78.5 kb of the α-globin gene region36. (f) RNA-DNA RASER-FISH image of a mouse erythroblast showing the α-globin genes detected with plasmid pA36 (red) against nascent α-globin transcripts54 (green) imaged by widefield deconvolution (maximum intensity projection). g-i, Immuno-RASER-FISH examples imaged by widefield deconvolution. g, Antibody detection of HP1α (red) combined with a plasmid probe pCx (green) (Brown 2018) detected in a DAPI-stained C127 cell nucleus (maximum intensity projection covering the central region). h, Immuno-RASER-FISH image of an antibody to fibrillarin (detecting nucleoli) (red) combined with a plasmid probe pCx36 (green) detected in a DAPI-stained C127 cell nucleus. i, Immuno-RASER-FISH image of an antibody to 53BP1 (red) combined with a BAC RP11-347N18 probe partly covering the KIF23 TAD38 (green) detected in a DAPI-stained U2OS cell nucleus (maximum intensity projection covering the central region). Scale bars are 5 µm and 1 µm (insets).</p