For immunostaining, eye sections were equilibrated to room temperature, washed, blocked, and incubated with the primary antibodies, goat anti m-leptin (R&D Systems, Minneapolis, MN, USA) and rabbit anti-C-peptide (Cell Signaling, Danvers, MA, USA) in the presence of 0.1% Triton X-100 and 10% serum. After washing, secondary antibodies, anti-goat Alexa633, and anti-rabbit Alexa594, respectively (Thermo Fisher Scientific, Waltham, MA, USA), were applied, and mounting with medium containing DAPI for nuclear counterstaining (Thermo Fisher Scientific, Waltham, MA, USA) was performed after repeated washing. Imaging was performed using a confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems, Wetzlar, Germany) with the following settings: DAPI excitation 405 nm, detection 450–470 nm; ZsGreen excitation 488 nm, detection 500–525 nm; mCherry excitation 548 nm, detection 560–580 nm; Alexa 594 (C-peptide staining) excitation 594 nm, detection 600–620 nm and Alexa 633 (leptin staining) excitation 633 nm, detection 640–680 nm. To avoid spectral overlap imaging was performed using in-between-frames sequential imaging.
Rabbit anti c peptide
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-C-peptide is a primary antibody that recognizes the C-peptide region of the insulin prohormone. It is commonly used in immunoassays and Western blotting applications to detect and quantify C-peptide levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti glucagon, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Dapi for nuclear counterstaining, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Mouse anti brdu, by Agilent Technologies (1 mentions)
Guinea pig anti swine insulin, by Agilent Technologies (1 mentions)
Rabbit anti survivin, by Cell Signaling Technology (1 mentions)
Biotinylated secondary antibody, by Zymo Research (1 mentions)
Peroxidase conjugated streptavidin, by Zymo Research (1 mentions)
Neo scmos monochrome camera, by Oxford Instruments (1 mentions)
Rabbit anti insulin, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti somatostatin, by Agilent Technologies (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Oct mounting medium, by Avantor (1 mentions)
Anti rat cd3, by BioLegend (1 mentions)
Rat anti cd8, by BioLegend (1 mentions)
Prolong diamond anti fade solution, by Thermo Fisher Scientific (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
7 protocols using rabbit anti c peptide
1
Immunostaining of Eye Sections
2
Immunohistochemical Analysis of Pancreatic Markers
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The paraffin sections were stained as previously described [12] (link). Antibodies used were guinea pig anti-swine insulin (Dako Cytomation, Glostrup, Denmark), mouse anti-BrdU (Dako Cytomation), rabbit anti-survivin (Cell Signalling Technology), rabbit anti-C-peptide (Cell Signalling Technology, Danvers, MA, USA) and rabbit anti-ChromograninA (Dako Cytomation). Specificity of the survivin antiserum was confirmed by preabsorbtion with a survivin blocking peptide for 30 min (Cell Signalling Technology).
For light microscopy stainings the sections were incubated for 30 min at room temperature with a biotinylated secondary antibody (Zymed Laboratories, South San Francisco, CA, USA), rinsed and incubated with peroxidase-conjugated streptavidin (Zymed Laboratories). The sections were finally developed with 3-amino-9-ethyl-carbazole substrate (Thermo Scientific). For fluorescence microscopy, the secondary antibodies used were goat anti-guinea pig IgG (Alexa, A11076, Invitrogen, Paisley, UK), donkey anti-rabbit IgG (Alexa, A21206, Invitrogen) and donkey anti-mouse IgG (Alexa, A21202, Invitrogen). Nuclear staining was performed with DAPI (Vectashield with DAPI, Vector Laboratories, Burlingame, CA, USA).
For light microscopy stainings the sections were incubated for 30 min at room temperature with a biotinylated secondary antibody (Zymed Laboratories, South San Francisco, CA, USA), rinsed and incubated with peroxidase-conjugated streptavidin (Zymed Laboratories). The sections were finally developed with 3-amino-9-ethyl-carbazole substrate (Thermo Scientific). For fluorescence microscopy, the secondary antibodies used were goat anti-guinea pig IgG (Alexa, A11076, Invitrogen, Paisley, UK), donkey anti-rabbit IgG (Alexa, A21206, Invitrogen) and donkey anti-mouse IgG (Alexa, A21202, Invitrogen). Nuclear staining was performed with DAPI (Vectashield with DAPI, Vector Laboratories, Burlingame, CA, USA).
3
Multicolor Immunostaining of Islet Cells
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Immunofluorescence labeling of islets from female C57BL/6J was performed to visualize colocalization of the probe in insulin positive glucagon negative cells. The intact pancreas was incubated with EP12-BTMR-X (37 °C, 30 min, 50 nM) in RPMI supplemented with 10% FCS and then washed twice with cold PBS. The pancreas was embedment in OCT compound (Sakura Finetek) and flash-frozen in an isopentane bath on dry ice. The frozen tissues were sectioned (5-um thickness), mounted on microscope slides, and stored at –80 °C. Slides were then incubated at 4 °C o/n with primary antibodies diluted with 4% goat normal serum in PBS solution. The following primary antibodies were used for immunostaining: rabbit anti-insulin (1:25, Santa Cruz), rabbit anti-glucagon (1:1000, Millipore) rabbit anti-somatostatin (1:250, Dako) and rabbit anti-c-peptide (1:100, Cell Signaling). A species-matched secondary antibodies were used for immune detection (Alexa Fluor 488 conjugated goat anti-rabbit, 1:100, Life Technology). Nuclear counterstaining was performed using DAPI (Invitrogen). Immunofluorescence images were acquired with an epifluorescence microscope, BX63 (Olympus), with a Neo sCMOS Monochrome camera (ANDOR).
4
Immunohistochemical analysis of mouse pancreas
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Livers and pancreata removed from sacrificed mice were fixed in 4% PFA for 5 h at 4 °C, dehydrated in 30% sucrose overnight at 4 °C, and embedded in O.C.T mounting medium (VWR Chemicals) before freezing at −70 °C. Five micrometre cryostat (Leica) sections were air-dried, fixed in acetone for 6 min, air-dried, and stored at −20 °C. Immunolabelling was performed on sections pre-blocked with 1% BSA by incubating with rabbit anti glucagon (Millipore, cat no. AB932, 1:20) guinea pig anti-insulin (DAKO, cat no. A0564, 1:150), rabbit anti C-peptide (Cell Signalling, cat no. 4593S, 1:50), Rat anti-CD3 (Biolegend, cat no. 100202, 1:100), Rat anti-CD8 (Biolegend, cat no. 100702, 1:50) for 1 h at 4 °C. Incubation with relevant Alexa Fluor® secondary antibodies (Invitrogen, 1:500) and DAPI (4′,6-diamidino-2-phenylindole) (Invitrogen) followed. Sections were then mounted in Prolong Diamond Antifade solution (Thermo Fisher Scientific) and visualised using a Zeiss LSM 700 confocal microscope (Zeiss, Germany). Images were captured and processed using Zen software (Zeiss).
5
Immunostaining of Pancreatic Progenitor Cells
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IPCs were fixed in 4% paraformaldehyde in PBS for 20 min at room
temperature. The cells were blocked for 1 hour at room temperature with
10% normal goat serum in PBS containing 0.1% Triton X-100. The
cells were stained with primary antibodies and Alexa Fluor 488 or Alexa 594
nm-conjugated secondary antibodies in PBS containing 1% BSA and
0.3% Triton X-100. The nuclei was stained with DAPI. The following
antibodies and dilutions were used : 1:100; mouse anti-pancreatic and duodenal
homeobox 1 (PDX1) (Santa Cruz, Dallas, TX, USA), 1:100; rabbit anti-c-peptide
(Cell signaling, Danvers, MA, YSA), 1:100, goat anti-Nkx6.1 (Santa Cruz), 1:100;
TRITC-conjugated goat anti-rabbit IgG (Sigma), anti-goat IgG (Alexa fluorescence
488) (Abcam, Cambridge, UK), goat anti-mouse IgG (Alex fluorescence 594) (Abcam,
Cambridge, UK) and goat anti-mouse (Alexa fluorescence 488) (Abcam, Cambridge,
UK).
temperature. The cells were blocked for 1 hour at room temperature with
10% normal goat serum in PBS containing 0.1% Triton X-100. The
cells were stained with primary antibodies and Alexa Fluor 488 or Alexa 594
nm-conjugated secondary antibodies in PBS containing 1% BSA and
0.3% Triton X-100. The nuclei was stained with DAPI. The following
antibodies and dilutions were used : 1:100; mouse anti-pancreatic and duodenal
homeobox 1 (PDX1) (Santa Cruz, Dallas, TX, USA), 1:100; rabbit anti-c-peptide
(Cell signaling, Danvers, MA, YSA), 1:100, goat anti-Nkx6.1 (Santa Cruz), 1:100;
TRITC-conjugated goat anti-rabbit IgG (Sigma), anti-goat IgG (Alexa fluorescence
488) (Abcam, Cambridge, UK), goat anti-mouse IgG (Alex fluorescence 594) (Abcam,
Cambridge, UK) and goat anti-mouse (Alexa fluorescence 488) (Abcam, Cambridge,
UK).
6
Ultrathin ME Sections Immunolabeling Protocol
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Ultrathin ME sections were collected on 300-mesh nickel grids as described previously (71 (link)). Grids were etched for 5 seconds in sodium ethanolate (a saturated solution of NaOH in 100% ethanol) at least 24 hours before use. Grids were rinsed in TBS with 0.1% Triton X-100 (TBST; pH 7.4), then incubated for 20 minutes in 2% human serum albumin (MilliporeSigma) containing 0.1% sodium borohydride and 50 mM glycine (pH 7.4). Grids were then washed in TBST and incubated for 3 hours at RT in a mixture of goat anti-mCRH (1:80; Santa Cruz Biotechnology) and rabbit anti–C-peptide (1:30; Cell Signaling Technology) antibodies. Grids were rinsed in TBST, incubated with 12 nm gold–conjugated donkey anti–goat IgG antibody (1:25 in TBST containing 0.05% polyethylene glycol; Jackson ImmunoResearch, 705-205-147) for 2 hours, rinsed in TBST, and incubated with 30 nm gold–conjugated goat anti–rabbit IgG antibody (1:25 in TBST containing 0.05% polyethylene glycol; BBI Solutions, EM.GAR30) for 2 hours. After rinsing in distilled water, the grids were stained with uranyl acetate and lead citrate and examined on a Hitachi H-7500 electron microscope at 80 kV accelerating voltage. Images were captured with Digital Montage software driving a MultiScan cooled ES1000W Erlangshen CCD Camera (Gatan, Inc.) attached to the microscope.
7
Immunostaining of 3D hAEC Spheroids
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3D hAEC spheroids were fixed with 2% Paraformaldehyde (PFA, Sigma-Aldrich) for 20 min at room temperature. After washing 2 × 10 min in PBS, spheroids were permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) in PBS for 10 min and then rinsed again 2 × 10 min in PBS. Antigen blocking was performed for 1 h at room temperature in blocking solution: 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA), 0.2% Triton X-100, 0.1% Bovine Serum Albumin (BSA) (Sigma-Aldrich). Primary and secondary antibodies were diluted in freshly prepared blocking solution. Primary antibodies were incubated at 8°C overnight and secondary antibodies for 40-50 min at room temperature. The following primary antibodies and dilutions were used: mouse anti-insulin 1:100 (Abcam ab46707); rabbit anti-glucagon (Abcam ab8055); rabbit anti-C-peptide 1:100 (Cell Signalling #4593); rabbit anti-CK19 1:100 (Thermo Scientific PA5-16726); mouse antilaminin 1:100 (Thermo Scientific MA1-21194). Secondary antibodies were: DyLight 594 goat antimouse 1:200 (Jackson Immuno Research Labs.); Alexa Fluor 488 goat anti-rabbit 1:400 (Molecular Probes); CF 488 goat anti-mouse 1:400 (Biotium); DyLight 594 goat anti-rabbit 1:200 (Thermo Scientific). Nuclei counterstaining was performed with Fluoroshield+DAPI (Sigma-Aldrich). Imaging
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