Differentiated cells were dispersed into single cells using trypsin 0.25% (Life Technologies) at 37C for 5 minutes and neutralized with DMEM 10% FBS (Logan, Utah). Single cells were isolated using 40 μM filters. The cells were fixed with 4% PFA for 20 minutes at RT, washed with ice-cold FACS buffer (PBS + 5% FBS and resuspended in FACS buffer). For cells stained for intracellular proteins, resuspension was done in FACS buffer containing 0.1% Triton X-100. Blocking and antibody staining were performed in 96-well plates. After 1 hour of blocking at RT, cells were subsequently resuspended in FACS buffer with primary antibodies and incubated for 1 hour at RT. Antibodies used: PDX1, ab47308 (Abcam) (1:100) and NKX6.1, LS-C124275 (Life Span, Seattle, Washington) (1:100). Cells were washed three times with FACS buffer and then incubated in FACS buffer with secondary antibodies for 1 hour at RT. Secondary antibodies used were Alex Fluor 488 Donkey Anti-Goat IgG (H+L), A11055 (1:500) and Alex Fluor 594 Goat Anti-Guinea Pig IgG (H+L), A11076 (1:500) (Thermo Fisher Scientific). After washing three times with FACS buffer, cells were analyzed using LSR-II flow cytometer (BD Biosciences, San Jose, California). FACS analysis was performed using FlowJo software.
Ab47308
Manufactured by Abcam
Ab47308 is a primary antibody that recognizes the target protein. It is suitable for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
G2654, by Merck Group (2 mentions)
A056401 2, by Agilent Technologies (2 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Dmem 10 fbs, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
E cadherin, by BD (1 mentions)
Gtx16667, by GeneTex (1 mentions)
Tissue tek optimal cutting compound, by Sakura Finetek (1 mentions)
Af1657, by R&D Systems (1 mentions)
Ihc 00352, by Fortis Life Sciences (1 mentions)
Nbp2 15339, by Novus Biologicals (1 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Cell fixing kit, by Funakoshi (1 mentions)
Ab70328, by Abcam (1 mentions)
Triton x 100, by Fujifilm (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Bz 9000, by Keyence (1 mentions)
7 protocols using ab47308
1
Intracellular Protein Staining of Differentiated Cells
2
Immunohistochemistry of Pancreatic Tissues
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We performed immunohistochemistry as described6 (link). We applied heart-perfused fixation with 4% paraformaldehyde (PFA). Pancreata were incubated in 4% paraformaldehyde (PFA) at 4 °C overnight, washed with ice-cold PBS three times, and placed in 30% sucrose overnight16 (link). Tissue was embedded in Tissue-Tek optimal cutting compound (Sakura Finetek), frozen on dry ice, and cut into frozen 5-μm sections. We applied antigen retrieval to detect transcription factors (Nacalai USA Inc.). We used primary antibodies to INSULIN (A056401-2; Dako; 1:1000), GLUCAGON (G2654; Sigma-Aldrich; 1:1000), PDX1 (ab47308; Abcam; 1:100), E-cadherin (61018; BD Biosciences; 1:100), REG1 (AF1657; R&D systems; 1:100), REG2 (AF2035; R&D systems; 1:100), REG3d (MAB5678; R&D systems; 1:100), NR5a2 (PPH2325; R&D systems; 1:100), KI67 (GTX16667; Genetex; 1:100), MAFA (IHC-00352; Bethyl Laboratories; 1:100), Cleaved Caspase-3 (9661; Cell Signaling; 1:100), and ALDH1A3 (NBP2-15339; Novus Biologicals; 1:100) and Alexa Fluor–conjugated goat serum as a secondary antibody (Jackson ImmunoResearch Laboratories and Molecular Probes). The images were captured using a Zeiss LSM 710 confocal microscope using a 20× objective and analyzed using ZEN.
3
Immunostaining of Differentiated iPSCs
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Immunostaining was performed on differentiated iPSCs as previously reported [32 (link), 36 (link)]. Cells were washed once with PBS then 4% paraformaldehyde (PFA) was added on the cells for 20 min and placed on a shaker at room temperature. The cells were then washed with tris-buffered saline + 0.5% Tween 20 (TBST) thrice in a 10-minute interval on a shaker. Cells were then permeabilized for 15 min at room temperature using phosphate buffered saline (PBS) + 0.5% Triton X-100 (PBST) twice, later blocked overnight with 6% Bovine Serum Albumin (BSA) in PBST at 4oC. Afterwards, guinea pig anti-PDX1 (1:500, ab47308, Abcam) and mouse anti-NKX6.1 (1:2000, F55A12-C, DSHB) primary antibodies diluted in 3% BSA in PBST were added to the cells and incubated overnight at 4oC. Cells were washed three times with TBST and then Alexa Fluor secondary antibodies (ThermoFisher Scientific) diluted in PBS (1:500) were added for 1 h at room temperature then washed again three times using TBST. Cell nuclei were stained for two minutes with Hoechst 33,258 (DAPI) diluted 1:5000 in PBS (Life Technologies, USA). After washing three times with PBS, images were captured using inverted fluorescence microscope (Olympus).
4
Immunohistochemical Analysis of Transplanted Islet Cells
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Generated cells and extirpated organs were prepared and stained by methods already reported12 (link). Briefly, generated IPCs were fixed using a cell fixing kit (Funakoshi), recipient mice were sacrificed and IPCs bearing kidneys were extirpated. 4 μm thick paraffin embedded sections were incubated with primary antibodies against insulin (aa287–299, LS-B129; LSBio) at a dilution of 1:100 in phosphate-buffered saline (PBS), anti-HLA class I (ab70328; abcam) 1:100 in PBS, C-peptide (bs-0274R, Funakoshi) 1:100 in PBS and pancreatic and duodenal homeobox 1 (PDX-1, ab47308;abcam) 1:200 in PBS for 1 h at room temperature after deparaffinization and antigen retrieval. Slides were then incubated with biotinylated secondary antibody, followed by treatment with a streptavidin-biotin-horseradish peroxidase complex. Positive staining was visualized with diaminobenzidine and cell nuclei were counterstained with Mayer’s haematoxylin.
5
Immunofluorescent Localization of PDX1
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Cells were fixed with 4% paraformaldehyde for 30 min at room temperature and then treated successively with 0.3% Triton X-100 (Wako Chemical; Osaka; Japan) in phosphate-buffered saline (PBS; Sigma) for 15 min followed by 3% bovine serum albumin (Sigma) for 30 min to reduce nonspecific reactions. Samples were allowed to react overnight with anti-PDX1 (Abcam ab47308) antibodies at a 1:200 dilution at 4°C. They were then stained with Alexa Fluor 488 conjugated anti-guinea pig IgG antibody (ab150185; Abcam, Tokyo, Japan) as the secondary antibody for 1 h at room temperature. Their nuclei were stained with DAPI for 10 min. Images were acquired on a fluorescence microscope (Keyence BZ-9000).
6
Single-cell immunophenotyping of pancreatic cells
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Cell clumps were dissociated into single cells using TrypLETM Express (Life Technologies) at 37 °C and passed through a 40 μm cell strainer. Single cells were fixed with 4% paraformaldehyde on ice for 30 min, then blocked in FACS buffer (5% FBS in DPBS) with (for intracellular protein) or without (for cell surface protein) 0.1% Triton X-100 on ice for 1 h, followed by incubation with primary antibodies overnight at 4 °C. The primary antibodies used were for the detection of HNF1A (Abcam, ab96777; 1:100), GATA4 (Thermo Fisher Scientific, MA5-15532; 1:100), PDX1 (Abcam, ab47308; 1:100), INS (Abcam, ab7842; 1:100) and NKX6.1 (LifeSpan BioSciences, LS‑C124275; 1:100). Cells were washed twice with FACS buffer and incubated with secondary antibodies in the dark at 4 °C for 1 h. The secondary antibodies used were Alexa Fluor® 488 (Invitrogen, A21202; 1:1000), Alexa Fluor® 488 (Invitrogen, A21206; 1:1000), Alexa Fluor® 647 (Invitrogen, A21447; 1:2000) and Alexa Fluor® 647 (Jackson ImmunoResearch, 706-605-148; 1:2000). The cells were then washed in FACS buffer twice, washed again in DPBS twice and resuspended in DPBS and analyzed with the BDTM LSR II Flow Cytometer. Data analysis was performed using the FlowJo 7.0 software.
7
Immunostaining of Pancreatic Cell Markers
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Cells were cultured on glass coverslips and fixed in 2% paraformaldehyde (PFA) for 15 min. The immunofluorescence protocol performed conformed to indications provided by the supplier. The following primary antibodies were used: mouse anti-β-catenin (1/500, ab22656, Abcam), rabbit anti-BMP4 (1/500, ab39973, Abcam), rabbit anti-ROR2 (1/50, ab92379, Abcam), rabbit anti-c-JUN (1/500, ab31419, Abcam), mouse anti-insulin (1/500, I2018, Sigma-Aldrich), guinea-pig anti-porcine insulin (1/500, A056401-2, Dako), mouse anti-porcine glucagon (1/500, G2654, Sigma-Aldrich), rat anti-somatostatin (1/100, sc-47706, Santa Cruz), guinea-pig anti-PDX1 (1/500, ab47308, Abcam), and rabbit anti-NKX6.1 (1/100, NBP1-82553, Novus), rabbit anti-MAFA (1/200, ab98859, Abcam). The following secondary antibodies were used: donkey anti-rabbit A488, donkey anti-rabbit A546, goat anti-mouse A568, chicken anti-rat A647, and goat anti-guinea-pig A647 (1/500, Molecular Probes). The nuclei were stained with DAPI (D1306, Molecular Probes). The samples were mounted in Prolong Diamond Antifade Mountant Media (P36970, Life technologies) and were analyzed with Leica TCS SP5 STED CW confocal microscope. No specific feature of the original data was obscured, eliminated or misrepresented.
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