Cells were fixed and immunostained with a standard protocol [15 (link)]. Antibodies used were: rabbit anti-Oct4 antibody (1/500; Santa Cruz; sc-9081), goat anti-Sox17 antibody (1/300; R&D; AF1924), mouse anti-Nkx6.1 (1/200; Developmental Studies Hybridoma Bank; F55A10), goat anti-Pdx1 (1/300; R&D; AF2419), mouse anti-Ngn3 (1/300; Developmental Studies Hybridoma Bank; F25A1B3), mouse anti-Insulin (1/600; Sigma-Aldrich; I2018), mouse anti-Glucagon (1/600; Sigma-Aldrich; G2654), rat anti-C-peptide (1/600; Developmental Studies Hybridoma Bank; GN-ID4), anti-rabbit IgG, Alexa Fluor 488 conjugated (1/600; Invitrogen; A21206), anti-goat IgG, Alexa Fluor 594 conjugated (1/600; Invitrogen; A11058), anti-mouse IgG, Alexa Fluor 488 conjugated (1/600; Invitrogen; A21202), anti-rat IgG, Alexa Fluor 594 conjugated (1/600; Invitrogen; A-11007). Nuclei were counterstained with DAPI (1/200; Dojindo; 340–07971) for 2D cultured cells and with TO-PRO3 (Thermo Fisher Scientific) for 3D cultured cells before specimens were mounted in Prolong Gold Antifade Reagent (Invitrogen). Immunostained specimens were examined with an inverted fluorescent microscope (Keyence, Japan) and confocal laser scanning microscope (OLYMPUS, Japan).
Inverted fluorescent microscope
Manufactured by Keyence
Sourced in Japan
The Inverted Fluorescent Microscope is a type of microscope that illuminates and observes samples from below. It is designed to view cells and other biological specimens in a culture dish or microplate. The microscope uses fluorescence to visualize specific cellular structures or molecules that have been labeled with fluorescent dyes or proteins.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Af2419, by R&D Systems (1 mentions)
Af1924, by R&D Systems (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
Sc 9081, by R&D Systems (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
Goat anti pdx1, by R&D Systems (1 mentions)
Mouse anti glucagon, by Merck Group (1 mentions)
Goat anti sox17 antibody, by R&D Systems (1 mentions)
Rabbit anti oct4 antibody, by Santa Cruz Biotechnology (1 mentions)
Rat anti c peptide, by Developmental Studies Hybridoma Bank (1 mentions)
2 protocols using inverted fluorescent microscope
1
Immunostaining of Stem Cell Markers
2
Immunofluorescence Staining of Pluripotent and Endocrine Markers
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Cells were fixed and immunostained with a standard protocol (Ninomiya et al. 2014 (link)). Antibodies used were listed as follows: rabbit anti-OCT4 antibody (1/500; Santa Cruz, Dallas, TX; sc-9081), goat anti-SOX17 antibody (1/300; R&D, Minneapolis, MN; AF1924), goat anti-PDX1 (1/300; R&D; AF2419), mouse anti-NGN3 (1/300; Developmental Studies Hybridoma Bank, Iowa City, IA; F25A1B3), mouse anti-GLUCAGON (1/600; Sigma-Aldrich, St. Louis, MO; G2654), rat anti-C-peptide (1/600; Developmental Studies Hybridoma Bank; GN-ID4), anti-rabbit IgG, Alexa Fluor 488 conjugated (1/600; Invitrogen; A21206), anti-goat IgG, Alexa Fluor 594 conjugated (1/600; Invitrogen; A11058), anti-mouse IgG, Alexa Fluor 488 conjugated (1/600; Invitrogen; A21202), anti-rat IgG, Alexa Fluor 594 conjugated (1/600; Invitrogen; A-11007). Nuclei were counterstained with DAPI (1/200; Dojindo, Kumamoto, Japan; 340–07971) before specimens were mounted in Prolong Gold Antifade Reagent (Invitrogen). Specimens were observed with an inverted fluorescent microscope (Keyence, Osaka, Japan).
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