Cells were dissociated using 1 × TrypLE Select Enzyme and washed with cold FACS buffer (5% FBS in 1 × DPBS). Cells were fixed with 4% paraformaldehyde and permeabilized with donkey block solution (0.1% tween-20, 10% FBS, 0.1% BSA and 3% donkey serum) containing 0.5% saponin. The cells were incubated with goat anti-SOX17 antibody (1:500, #GT15094, Acris/Novus) and goat anti-PDX1 antibody (1:500, #AF2419, R&D Systems) for 30 min at room temperature and then stained with Alexa Fluor 555-conjugated donkey antibody directed against goat (1:500, #A21432, Invitrogen) for 30 min at room temperature. Flow cytometry was performed using FACS-Aria III (BD Bioscience). FACS data were analyzed using FlowJo.
Af2419
Manufactured by R&D Systems
AF2419 is a lyophilized recombinant human Follistatin-related Protein 1 (FSTL1) protein. FSTL1 is a secreted glycoprotein that is involved in the regulation of cell growth and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Af1924, by R&D Systems (4 mentions)
Af2400, by R&D Systems (3 mentions)
Axio observer microscope, by Zeiss (2 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (2 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Af2606, by Santa Cruz Biotechnology (2 mentions)
Sc 22840x, by R&D Systems (2 mentions)
Ab8895, by Abcam (2 mentions)
Facsaria 3, by BD (1 mentions)
A21432, by Thermo Fisher Scientific (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab14181, by Abcam (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
Sc 9081, by R&D Systems (1 mentions)
Inverted fluorescent microscope, by Keyence (1 mentions)
10 protocols using af2419
1
Multiparametric Flow Cytometry Analysis
2
Immunofluorescence Staining of Endoderm Markers
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The cells were fixed in 1% paraformaldehyde for 30 min. After washing 3 times with PBS, the cells were blocked and permeabilized in blocking solution (PBS containing 3% bovine albumin and 0.2% Triton X-100) for 30 min at room temperature. The cells were then incubated with primary antibodies in blocking solution at 4 °C overnight, washed 3 times, and incubated with the corresponding secondary antibodies for 1 h at room temperature. The cells were washed twice and stained with DAPI (Sigma) for 5 min and then analyzed using a Leica DMI6000B microscope (Leica Microsystems). Primary antibodies used in this study were as follows: FoxA2 (R&D, AF2400, 1:200), Sox17 (RD, AF1924, 1:200), goat anti-Pdx1 (R&D, AF2419, 1:200), guinea pig anti-insulin (Dako, A056401, 1:300), and rabbit anti-C-peptide (Abcam, ab14181, 1:300).
3
Immunostaining of Stem Cell Markers
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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).
4
Immunofluorescence of Ductal Organoids
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For immunofluorescence of the ductal organoids, 10.000 single cells were seeded in an 8 Chamber Well Slide coated with Matrigel-GFR (LAB-TEK, #440263 0903). After 5 days, the organoids were washed twice with PBS 1X and fixed with 2% buffered paraformaldehyde for 20 min at room temperature. Subsequently, the organoids were again washed 3 times with PBS 1X and then permeabilized with Triton 0.7% for 15 min at RT. After blocking for 1 hour at RT (normal goat serum 5%, BSA 1%, Triton 0.4%), the primary antibodies were incubated overnight at 4°C. The following antibodies were used: cytokeratin 19 (TROMA-III, DSHB), Ki-67 (MA5-14520, Invitrogen), SOX9 (AB5535, Millipore), FOXA2 (Ab108422, Abcam), and PDX1 (AF2419, R&D). Images were acquired on an Axioplan2 microscope (Carl Zeiss) equipped with an AxioCamHR camera and AxioVision Version 4.8 (both from Carl Zeiss) software. Magnifications are given in figure legends.
5
Immunofluorescence Staining of Differentiated Cells
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Differentiated cells on tissue culture plates were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at RT followed by washing with PBS. Permeabilization was performed with PBS with 0.2% Triton (PBST) by incubating for 30 min at RT. 5% donkey serum in PBST was used as a blocking solution and antibody staining solution. After blocking for 30 min at RT, blocking solution containing primary antibodies was added. The following primary antibodies and dilutions were used: goat anti-PDX1 (R&D, AF2419, 0.4 μg/ml), mouse anti-NKX6–1 (DSHB F55A12-c, 1:500). Primary antibodies in blocking solution were incubated overnight at 4°C. After washing with PBST, Alexa Fluor secondary antibodies (Molecular Probes, 1:500) were incubated at RT for 1 hour. DAPI (Sigma, 32670–5MG-F, 0.2 μg/ml) was used to stain cell nuclei. Zeiss Axio Observer microscope was used for imaging. Antibodies for this study are summarized in Table S8 .
6
Immunofluorescence Staining of Human ESCs
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Human ESCs differentiated on µ-Plate 24-wells (Ibidi) were used for in-well immunofluorescence staining. Cells were washed with PBS followed by fixation in 4% PFA solution for 20 min at RT. Subsequently, fixed cells were washed three times with PBS and background staining was quenched with 50 mM NH4Cl for 10 min. After washing with PBS, cells were incubated in PBS containing 0.1% Triton-X and 5% normal donkey serum (blocking) at room temperature for 45 min and subsequently, in blocking solution with primary antibodies overnight at 4 °C. Cells were washed twice with PBS containing 0.1% Triton-X and 2% normal donkey serum (wash solution) followed by incubation in blocking solution containing secondary antibodies at room temperature for 1.5 h. Finally, cells were washed with PBS and nuclei were stained with 500 ng/ml DAPI. Images were acquired on a Keyence Biozero BZ-9000 microscope. Antibodies used: OCT3/4 (Santa Cruz; sc-5279; 1:200), NANOG (Cell Signaling; #3580; 1:100), SOX17 (R&D; AF1924; 1:500), PDX1 (R&D; AF2419; 1:500), NKX6.1 (DSHB; F55A10 concentrate; 1:100) in combination with Alexa-conjugated secondary antibodies from Invitrogen.
7
Chromatin Immunoprecipitation with Dynabeads
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50 μL of Dynabeads (Thermo Fisher Scientific 10009D), which were previously blocked with 1% BSA, were added to samples and incubated at 4°C with rotation for 1 hour. After pre-clearing, Dynabeads beads were removed by spinning down at 4,000 rpm on 4°C for 5 min. The supernatant was transferred to new 15 mL falcon tubes and 200 μL out of 10 mL volume was collected as 2% input. Antibodies were added to the pre-cleared samples for overnight incubation at 4°C with rotation. The following antibodies were used: rabbit anti-H3K4me1 (Abcam, ab8895, 5 μg), rabbit anti-H3K27ac (active motif, 39133, 5 μg), goat anti-FOXA2 (R&D, AF2400, 10 μg), rabbit anti-GATA6 (Cell Signaling Technology, 5851, 10 μg), goat anti-GATA4 (R&D, AF2606, 10 μg), rabbit anti-HNF1B (Santa Cruz, sc-22840x, 10 μg), goat anti-PDX1 (R&D, AF2419, 10 μg). 200 μL of Dynabeads, which were previously blocked with 1% BSA, were incubated at 4°C for 6 hours with rotation. After bead incubation, chromatin-bound beads were collected by centrifugation at 3,000 rpm for 5 min at 4°C.
8
Immunofluorescence Staining of Differentiated Cells
9
Chromatin Immunoprecipitation with Dynabeads
10
Immunohistochemical Analysis of Endoderm Markers
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Spheres derived from MEL1 were fixed for 15 min at RT with 4% paraformaldehyde, washed in PBS and subsequently embedded in 2% agarose (Sigma). Samples were dehydrated and paraffin embedded, followed by sectioning at 5 μm thickness. For staining, rehydrated sections were treated with antigen retrieval solution (Biogenex), blocked (CAS-Block, Life Technologies with 0.2% Triton-X 100, Sigma) and incubated in primary antibodies at 4 °C overnight. After washing in PBS with 0.1% Tween20, sections were incubated with secondary antibodies for 45 min at room temperature. Finally, slides were washed in PBS-T and PBS and mounted with Vectashield. Images were acquired using a Zeiss ApoTome. The following antibodies were used: SOX17 (R&D; AF1924; 1:500), PDX1 (R&D; AF2419; 1:500), NKX6.1 (DSHB; F55A12 concentrate; 1:150), and Alexa-conjugated secondary antibodies from Invitrogen. Nuclei were visualized with DAPI.
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