custom fluorescence rnascope probes  (Advanced Cell Diagnostics Inc)

Journal: The FASEB Journal

Article Title: Regulator of G-protein signaling Gβ5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion

doi: 10.1096/fj.201700197RR

Figure Lengend Snippet: Gβ5 knockout inhibits M3R-stimulated insulin secretion. Cholinergic stimulation of insulin secretion was tested in primary islets (A, B) or MIN6 cells lacking Gβ5 (E). A) Islets from Gnb5−/− (KO, red) and wild-type (WT, black) mice were perifused. After equilibration with media containing 3 mM glucose (3G), islets were challenged with 16.7 mM glucose (16G), with or without 10 μM Oxo-M. The data points are the mean ± sd of insulin concentrations measured in collected fractions from experiments on 4 independent animal cohorts (3–5 animals/genotype). ***P < 0.001 in the peak; for other data points, P < 0.05. B) Quantification of total secreted insulin (area under the curve shown in A). C) Immunoblot analysis of islets from Gnb5−/− mice (top) and CRISPR-Cas9-knockout MIN6 clones (bottom). Total cell lysates were probed for Gβ5, Gβ1, Gαq, and actin (representative of at least 2 experiments). D) In situ RNA hybridization of mouse pancreatic slices using RNAscope probes against Gnb5 (green) and Chrm3 (red) gene products was performed. Shown are phase-contrast (left) and fluorescence (right) images of a representative islet. The inset shows magnification of the fluorescent dots representing the single target mRNA molecules. E) Wild-type MIN6 cells, control CRISPR (LZ), and 2 stable clones after CRISPR-Cas9-mediated knockout of the Gnb5 gene (#1 and #2) were stimulated with 16.7 mM glucose or 16.7 mM glucose plus 100 μM Oxo-M. Data are means ± sd of insulin concentration in the supernatant from the cultured cells. *P < 0.05; **P < 0.01.

Article Snippet: In situ mRNA hybridization Localization of mRNA in paraffin-embedded slices of the mouse pancreata and eyes was performed with custom fluorescence RNAscope probes (Advanced Cell Diagnostics, Newark, CA, USA), according to the manufacturer’s instructions, with minor modifications described earlier ( 27 ).

Techniques: Knock-Out, Western Blot, CRISPR, Clone Assay, In Situ, Hybridization, RNAscope, Fluorescence, Control, Concentration Assay, Cell Culture

Journal: Molecular Pharmacology

Article Title: Teaching an Old Drug New Tricks: Agonism, Antagonism, and Biased Signaling of Pilocarpine through M3 Muscarinic Acetylcholine Receptor

doi: 10.1124/mol.117.109678

Figure Lengend Snippet: Pilocarpine is a full agonist in the pupil-constriction assay. Mouse eyes were treated ex vivo with the indicated drugs, and pupil diameter was analyzed as described in Materials and Methods. (A) Photographs of the eyes after 1-hour incubation in 10 mM pilocarpine or 0.01 mM CCh. (B) Time-course of pupil constriction in the presence of 10 mM Pilo or 0.01 mM CCh. (C) Eyes were treated with indicated concentrations of pilocarpine, Oxo-M and CCh for 10 minute. Data show average ± S.D. from three independent experiments. Symbols on the x-axis denote estimated Kd values for pilocarpine (30 μM), Oxo-M (50 μM), and CCh (150 μM) (from Sykes et al., 2009). (D) In situ RNA hybridization of mouse-eye section was performed using RNAscope approach (see Materials and Methods); shown is a representative image. Distinct green fluorescent dots correspond to individual mRNA molecules.

Article Snippet: Localization of Chrm3 messenger RNA was done using a custom fluorescence RNAscope probe (Advanced Cell Diagnostics, Newark, CA).

Techniques: Ex Vivo, Incubation, In Situ, Hybridization, RNAscope