fluorescein conjugated streptavidin cy5  (Vector Laboratories)

Journal: Nucleic Acids Research

Article Title: Repeat-rich RNA guides repetitive genomic elements into biomolecular condensates for heterochromatin organization and muscle integrity

doi: 10.1093/nar/gkag168

Figure Lengend Snippet: CU-rich RNA promotes heterochromatin condensate organization during differentiation. ( A ) Nuclei of C2C12 myotubes (MT) treated with 1.5% 1,6-hexanediol (1,6-HD) or 1.5% 2,5-hexanediol (2,5-HD). Left: Representative images of 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Scale bar, 5 μm. Middle: Time-course quantification of the number of heterochromatin foci per nucleus. Right: Boxplots show foci area (μm 2 , y -axis) at 5, 10, and 15 min posttreatment ( x -axis). n = 55 nuclei, three biological replicates. ( B ) Representative live-cell images of Hoechst 33342-stained MT nuclei before and after 1,6-HD treatment (0 and 15 min, respectively), taken from . Arrows indicate changes in heterochromatin foci intensity (pink, increased; blue, decreased), and the red arrow highlights an alteration in chromocenter integrity. ( C ) Number of heterochromatin foci per nucleus in MT following recovery from 1.5% 1,6-hexanediol (1,6-HD) treatment for 15 min, measured at indicated time points. n = 68 nuclei, three biological replicates. ( D ) Representative images of nuclei of myoblast (MB) and MT with or without 1,6-HD treatment (1.5%, 15 min). Right: Quantification of the number of heterochromatin foci per nucleus. n = 40 (MB), n = 60 (MT), three biological replicates. ( E ) Quantification of colocalization between indicated proteins and DAPI foci in MT with or without 1,6-HD treatment (1.5%, 15 min), by Pearson’s correlation coefficients. n = 60, three biological replicates. ( F ) Boxplot showing the distribution of Z -score of the interchromosomal interaction frequencies in MB and MT. ( G ) RNA FISH using ChRO1 and LacZ biotinylated probes. Biotin signal was detected by Fluorescein-conjugated Avidin DCS and amplified with biotinylated anti-Avidin and additional Fluorescein Avidin DCS. Right: Quantification of colocalization between biotin signal and DAPI foci. n = 50 nuclei. ( H ) Number of heterochromatin foci per nucleus of mouse fibroblast cells (NIH3T3) with or without doxycyline (Dox)-induced ChRO1a expression and/or 1,6-HD treatment (1.5%, 15 min). (EV; empty vector). n = 50, three biological replicates. ( I ) Number of heterochromatin foci per nucleus in MB with or without Dox-induced ChRO1a fragment (1–413, CUR) expression and/or 1,6-HD treatment (1.5%, 15 min). n = 75, three biological replicates. Statistical analyses and data presentation details are described in the “Materials and methods” section.

Article Snippet: The cells were then incubated with Fluorescein Avidin DCS (5 μg/ml, Vector Laboratories, A-2011) for 30 min at RT.

Techniques: Staining, Avidin-Biotin Assay, Amplification, Expressing, Plasmid Preparation

Journal: Cells

Article Title: Deletion of the Murine Ortholog of the Human 9p21.3 Locus Leads to Insulin Resistance and Obesity in Hypercholesterolemic Mice

doi: 10.3390/cells13110983

Figure Lengend Snippet: Chr4 Δ70/Δ70 does not affect pancreatic β-cell number but may reduce the cell proliferation rate already in young Ldlr −/− ApoB 100/100 mice. ( A ) Pancreatic islet size, ( B ) proportion of α- and β-cells, and ( C ) proliferation of mixed and ( D ) >10,000 μm 2 pancreatic islets in young Ldlr −/− ApoB 100/100 ( n = 5–7) and Chr4 Δ70/Δ70 mice ( n = 5–7). Primary antibody against glucagon was used as a marker for the pancreatic α-cells, and insulin ab was used for the β-cells. For visualization, fluorescent secondary antibodies were used. In the representative figures, glucagon positive cells appear in red and insulin in green. Nuclei were counterstained with DAPI (blue). Ki-67 antibody was used as a marker of cell proliferation and visualized with DAB. ( E ) For the islet function, insulin secretion of Ldlr −/− ApoB 100/100 ( n = 4) and Chr4 Δ70/Δ70 mice ( n = 4) was measured both in fasted state (0 min) and in response to 1 g/kg i.p. glucose at time points 15, 30, 60 and 90 min after the administration. Asterisk (*) indicates statistical significance. Difference in mean between Ldlr −/− ApoB 100/100 and Chr4 Δ70/Δ70 mice was measured by using t -test, and it was considered statistically significant when p < 0.05.

Article Snippet: Pancreatic α- and β-cells were immunostained with primary antibodies against glucagon (Dako A0565, Rabbit anti-human glucagon, Agilent, Santa Clara, CA, USA) and insulin (Dako A0564, Guinea pig anti-insulin, Agilent, Santa Clara, CA, USA) and appropriate fluorescent secondary antibodies (for glucagon, A21442, chicken anti-rabbit A594, Thermo Fisher Scientific, Waltham, MA, USA, and for insulin, BA-7000 Goat anti-guinea pig with A-2011 Fluorescein Avidin DCS, Vector laboratories, Newark, CA, USA).

Techniques: Marker