Mouse ES cell-derived cells were fixed with methanol and blocked with 10% FBS. Cells were then incubated with the primary antibodies: anti-insulin (1 : 200; Cell Signaling Technology, Danvers, MA, USA) and anti-Mfn2 (1 : 100; Abcam, Cambridge, MA, USA) at 4°C overnight. The cells were treated with the appropriate secondary antibodies and DAPI (Sigma-Aldrich). The overlay images were merged using the Image-Pro Plus software.
Anti insulin
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-insulin is a laboratory tool used to detect and measure the presence of insulin in biological samples. It is a highly specific antibody that binds to insulin, allowing researchers to quantify insulin levels in cells, tissues, or body fluids.
Automatically generated - may contain errors
Lab products found in correlation
Anti pdx1, by Cell Signaling Technology (4 mentions)
Anti glucagon, by Cell Signaling Technology (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Ab239372, by Abcam (2 mentions)
Ab207750, by Abcam (2 mentions)
Anti ki67, by Abcam (2 mentions)
Anti foxo1, by Cell Signaling Technology (2 mentions)
Anti arid1a, by Abnova (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Anti mfn2, by Abcam (1 mentions)
Anti snap25, by Santa Cruz Biotechnology (1 mentions)
Anti pimt, by Abcam (1 mentions)
Anti mafa, by Cell Signaling Technology (1 mentions)
Anti hdac5, by Cell Signaling Technology (1 mentions)
Anti neurod1, by Cell Signaling Technology (1 mentions)
Anti brdu, by Agilent Technologies (1 mentions)
A11005, by Thermo Fisher Scientific (1 mentions)
Anti c peptide, by Cell Signaling Technology (1 mentions)
26 protocols using anti insulin
1
Immunofluorescence Detection of Insulin and Mfn2
2
Protein Expression Analysis in BRIN-BD11 Cells
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BRIN-BD11 cells were lysed using TENNS lysis buffer [120 mM of NaCl, 1% Triton X-100, 20 mM of Tris–HCl pH 7.5, 10% glycerol, 2 mM of EDTA and protease inhibitor cocktail (10 μg/mL each of aprotinin and leupeptin)]. Approximately 30 μg of protein was resolved by 10% SDS-PAGE and a Western blot analysis was carried out using specified antibodies. The catalogue and primary dilution of the antibodies used in the study are Anti-PIMT (Abcam, Cambridge, UK, catalogue no. Ab70559) 1:2000; anti-PDX1 (Cell Signaling Technologies, Danvers, MA, USA, catalogue no. 5679) 1:1000; anti-MafA (sc-390491) 1:200); anti-HDAC5 (sc-133225) 1:1000; anti-GCK (sc-17819) 1:200; anti-insulin (Cell Signaling Technologies, 8138) 1:1000; anti-Synaptotagmin13 (Novus Biologicals, NB2-20546) 1:1000; anti-SNAP25 (sc-390644 Santa Cruz Biotechnology, Finnell Street Dallas, TX, USA) 1:200; anti-Kir6.2 (sc-20809) 1:200; anti-NeuroD1 (Cell Signaling Technologies, 2833) 1:1000; and anti-GAPDH (Cell Signaling Technologies, 2118) 1:5000. The protein levels were normalized using GAPDH and Western blots were quantified by using ImageJ [32 (link)].
3
Immunoanalysis of Pancreatic β-Cells
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Pancreases were analyzed by immunolabeling using anti-BrdU (Dako) and anti-insulin (Cell Signaling Technology) antibodies. Quantification of β-cell mass was performed as described previously [4] (link), [6] (link). TRPV1 expression in the pancreas was analyzed by immunostaining using a rabbit anti-TRPV1 antiserum as described in [36] (link). DRGs were stained with antibodies for TRPV1 (Alomone Labs) and CGRP (Sigma).
4
Immunofluorescence Staining of Insulin
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Fixed pancreatic tissue sections were blocked with 3% bovine serum albumin (BSA) and 5% normal goat serum in PBS for 1 hr before incubating with the primary antibody (anti-insulin, 1:100, Cell Signalling) diluted in 3% BSA in PBS at 4°C overnight. After washing in PBS, the appropriate fluorescent secondary antibody was used (Alexa Fluor) in 3% BSA in PBS for 2 hr at room temperature. Sections were then washed in PBS before mounting with gel mount containing DAPI (VECTASHIELD). Images were taken using a Leica DM4000 B LED fluorescent microscope using a 40x objective, or a Leica DMi8 TCSP8 confocal using a 40x objective.
5
Histological Assessment of Pancreas and Liver
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Pancreas sections were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 5 μm sections, and rehydrated by xylene and declining grades of ethanol for 5 min. Then, they were washed three times for HE staining. Immunohistochemical and immunofluorescent tests were performed using anti-PDX-1 (Abcam, UK, ab47383) antibody and anti-insulin (Cell Signaling Technology, USA, #4590S) antibody. Frozen livers were sliced, and Oil Red O staining was performed as described earlier [30 (link), 31 (link)] using the Oil Red O Stain Kit (Lipid Stain) (SenBeiJia Biological Technology, China, BP-DL101). According to the instructions of the kit, the Oil Red O solution was added dropwise onto the tissue for a 5-10 min incubation period. Excess staining buffer was removed with 85% propylene glycol. The tissues were washed with distilled water and counterstained with hematoxylin.
6
Immunofluorescence Staining of Pancreatic Markers
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Immunofluorescence staining was performed as previously reported10 (link). The following primary antibodies were used: anti-insulin (dilution 1:100, #4590; Cell Signaling Technology), anti-C-peptide (dilution 1:100, #4593S; Cell Signaling Technology), anti-PDX-1 (dilution 1:200, #5679; Cell Signaling Technology), anti-NKX6.1 (dilution 1:400, #54551; Cell Signaling Technology), anti-ICA (dilution 1:500, ab207750; Abcam plc.), anti-ZnT8 (dilution 1:500, 16169–1-AP, Proteintech, Chicago, IL, USA), anti-GAD65 (dilution 1:50, ab239372, Abcam plc.) and anti-PD-L1 (dilution 1:25, 17952–1-AP, Proteintech). Anti-rabbit Alexa 488 (A-11008; Thermo Fisher Scientific Inc.) for anti-C-peptide, PDX-1, ICA, ZnT8, GAD65 and PD-L1 antibodies, and anti-mouse Alexa 594 (A-11005; Thermo Fisher Scientific Inc.) for anti-insulin antibody were used as the secondary antibodies. 4′, 6-diamidino-2-phenylindole (DAPI) (P-36931; Thermo Fisher Scientific Inc.) was applied for nucleus staining.
7
Immunophenotyping of Differentiated Cells
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Mouse ES or human ES differentiated cells at terminal differentiation day were digested into single cells with Accutase. After being fixed in 4% paraformaldehyde for 1 h at 4 °C, cells were blocked with 3% BSA for another 1 h at room temperature. Then, the cells were incubated at 4 °C overnight with primary antibodies: anti-insulin (1 : 200, Cell Signaling Technology), anti-PPARα (1 : 200, Abcam, Cambridge, MA, USA), anti-PPARβδδ (1 : 200, Abcam) or anti-PPARγ (1 : 200, Abcam). After that, cells were incubated with the appropriate secondary antibodies (1 : 500) for 20 min at 4 °C. Cells were collected with a FACS flow cytometer (Beckman Coulter, Carlsbad, CA, USA). The results were expressed as the percentage of the fluorescence intensity.
8
Comprehensive Immunohistochemical Profiling of Pancreatic Cells
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Immunohistochemical staining was performed as previously reported56 (link) with the following antibodies: anti-insulin (dilution 1:100, #4590; Cell Signaling Technology), anti-PDX-1 (dilution 1:50, #5679; Cell Signaling Technology), anti-PD-L1 (dilution 1:100, 17952–1-AP, Proteintech), anti-ICA (dilution 1:500, ab207750; Abcam plc.), anti-ZnT8 (dilution 1:500, 16169–1-AP, Proteintech), anti-GAD65 (dilution 1:2000, ab239372; Abcam plc.), anti-CD4 (dilution 1:1000, ab183685; Abcam plc.) and anti-CD8 (dilution 1:2000, ab217344; Abcam plc.).
9
Immunohistochemical Analysis of Pancreatic Insulin and CD45
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Pancreatic sections were deparaffinized, blocked, and loaded with anti-insulin (Cell Signaling, 1:400) or anti-CD45 (1:200) antibodies as described in section 2.10 . The slides were loaded with anti-rabbit POD conjugate (TaKaRa) and incubated for 30 min at room temperature. The slides were then loaded with DAB substrate solution (Nichirei) and incubated for 30 min at room temperature. The slides were washed with water, counterstained with hematoxylin, dehydrated, and mounted in malinol (Muto). Images were taken using a BZ-X810 fluorescence microscope (Keyence).
10
Immunofluorescent Analysis of Pancreatic Islets
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Pancreatic tissue sections were incubated with primary antibodies overnight at 4 °C. The primary antibodies were as follows: anti-Glucagon (Cell Signaling Technology, Cat#2760), anti-insulin (Cell Signaling Technology, Cat#3014). The sections were stained with secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit IgG, Abcam, Cat#ab150077) for 2 h at 4 °C in dark. After washing with PBS, the sections were stained with DAPI and observed by confocal microscopy (Leica TCS SP8 STED 3X).
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