G2654

Manufactured by Merck Group

Sourced in United States, Denmark, United Kingdom

The G2654 is a laboratory equipment product manufactured by Merck Group. It serves as a general-purpose instrument for various scientific applications. The core function of the G2654 is to provide controlled environmental conditions for conducting experiments and analyses. Detailed specifications and intended use are not included in this concise factual description.

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Lab products found in correlation

A0564, by Agilent Technologies (15 mentions) Lsm 710 confocal microscope, by Zeiss (6 mentions) Ab30788, by Abcam (4 mentions) A056401 2, by Agilent Technologies (3 mentions) A 11076, by Thermo Fisher Scientific (3 mentions) Ab15580, by Abcam (3 mentions) P36931, by Thermo Fisher Scientific (3 mentions) D1306, by Thermo Fisher Scientific (3 mentions) Sc 10368, by Santa Cruz Biotechnology (2 mentions) Alexa 405, by Thermo Fisher Scientific (2 mentions) Ab28364, by Abcam (2 mentions) Tcs spe, by Leica (2 mentions) Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (2 mentions) Gw10064f, by Merck Group (2 mentions) A0565, by Agilent Technologies (2 mentions) A0566, by Agilent Technologies (2 mentions) Ab152, by Merck Group (2 mentions) A0010, by Agilent Technologies (2 mentions) Ab92547, by Abcam (2 mentions) Ab13970, by Abcam (2 mentions)

37 protocols using g2654

Dual Immunofluorescence for NBC1, Amylase, Insulin, and Glucagon

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The paraffin sections were deparaffinized in xylene, then rehydrated in graded ethanol and distilled water. The nonspecific binding sites were blocked in 1% BSA for 30 min. For NBC1 and amylase, insulin, glucagon double immunofluorescence, the rabbit anti-NBC1 primary polyclonal antibodies were applied and revealed using the FITC-labeled donkey anti-rabbit IgG (AP-182F, diluted 1:200; Chemicon, Inc). Mouse anti-amylase primary polyclonal antibody (sc-45667, diluted 1:500; Santa Cruz Biotechnology, Inc, Santa Cruz, CA, United States), mouse anti-insulin primary monoclonal antibody (ab6995, diluted 1:2000; abcam, Cambridge, United Kingdom) or mouse anti-glucagon primary polyclonal antibody (G-2654,diluted 1:2500; Sigma Co, St. Louis, MO, United States) was then applied and revealed by CY3-labeled donkey anti-mouse IgG (AP192C, diluted 1:400; Chemicon Inc). Sections were placed in Mounting Medium (Gel Mount Aqueous, G0918; Sigma Co.) with a cover glass and were examined under an Olympus BX51 Research Microscope. To rule out cross-reactivity in this staining system, the controls used were: first, single staining with the alternative secondary antibody; and second, staining in the absence of primary antibody. In neither case was staining detectable. Images were acquired at × 400 magnification.

Cao L.H., Xia C.C., Shi Z.C., Wang N., Gu Z.H., Yu L.Z., Wan Q, & De W. (2016). Na+/HCO3- cotransporter is expressed on β and α cells during rat pancreatic development. World Journal of Gastroenterology, 22(43), 9525-9533.

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Detailed Immunofluorescence Staining Protocol

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Samples processes for immunofluorescence were performed as described previously ^{6 (link)}. Frozen sections were cut at 10 μm-thick. Primary antibodies used were: guinea pig anti-porcine insulin (A0564, DAKO, 1:600), chicken anti-insulin (GW10064F, Sigma, 1:1000), mouse anti-glucagon (G2654, Sigma, 1:1000), rabbit anti-glucagon (A0565, DAKO, 1:600), rabbit anti-somatostatin (A0566, DAKO, 1:600), rabbit anti-pancreatic polypeptide (T-4088, PenLabs, 1:750), goat anti-ghrelin (sc-10368, SantaCruz, 1:200), rabbit anti-GFP (Life Technologies, 1:500), chicken anti-GFP (ab-13970, Abcam, 1:500), guinea pig anti-Pdx1 (gift from C. Wright, 1:1000), rabbit anti-MafA (A300–611A, Bethyl, 1:500), rabbit anti-Nkx6.1 (BCBC, 1:400), rabbit anti-pHH3 (06–570, Upstate, 1:500), rabbit anti-CD31 (ab28364, abcam, 1:50), rabbit anti-Vimentin (ab92547, abcam, 1:100), rabbit anti-Tyrosine hydroxylase (ab152, chemicon, 1:1000), guinea pig anti-ARX(AB2834, BCBC, 1:100), rabbit anti-Synaptophysin (A0010, DAKO, 1:50). Secondary antibodies were coupled to Alexa 405, 488, 568, 647 (Life Technologies), FITC, Cy3, Cy5 (Jackson Immunoresearch), or TRITC (Southern Biotech). Sections were counterstained with DAPI. All sections were examined with a confocal microscope (Leica TCS SPE). In Fig. 2i, confocal tile-scan images were merged as a maximum projection.

Furuyama K., Chera S., van Gurp L., Oropeza D., Ghila L., Damond N., Vethe H., Paulo J.A., Joosten A.M., Berney T., Bosco D., Dorrell C., Grompe M., Ræder H., Roep B.O., Thorel F, & Herrera P.L. (2019). Diabetes Relief in Mice by Glucose-Sensing Insulin-Secreting Human α-Cells. Nature, 567(7746), 43-48.

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Immunohistochemistry of Pancreatic Tissues

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We performed immunohistochemistry as described^{6 (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 overnight^{16 (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.

Son J., Du W., Esposito M., Shariati K., Ding H., Kang Y, & Accili D. (2023). Genetic and pharmacologic inhibition of ALDH1A3 as a treatment of β-cell failure. Nature Communications, 14, 558.

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Immunostaining of Pancreatic Tissues

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Formalin-fixed pancreas sections underwent antigen retrieval in boiling citrate buffer (pH 6.0) for 10 min before labeling with antibodies against insulin (catalog no. A0564; DAKO), glucagon (G2654; Sigma), and DAPI (P-36931; Invitrogen).

Ren D., Sun J., Wang C., Ye H., Mao L., Cheng E.H., Bell G.I, & Polonsky K.S. (2014). Role of BH3-Only Molecules Bim and Puma in β-Cell Death in Pdx1 Deficiency. Diabetes, 63(8), 2744-2750.

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Immunofluorescence Analysis of Expanded Islet Clusters

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Expanded islet clusters were harvested using cell recovery solution (Corning, 354253) and fixed in 4% paraformaldehyde for 30 min at room temperature, followed with blocking and permeabilizing in PBS with 0.5% Triton X-100 (Solarbio, T8200) and 5% donkey serum (Solarbio, SL050) for 30 min at room temperature. Then, samples were incubated with primary antibody at 4 °C overnight, followed by incubation with secondary antibody for 2 h at room temperature. DAPI (Beyotime, C1002) was used to stain the nucleus and find islets. The following antibodies were used for immunofluorescence: anti-insulin (1:200, sc-9168; Santa), anti-somatostatin (1:600, ab30788; Abcam), anti-glucagon (1:200, G2654; Sigma). anti-PDX1 (1:200, ab47267; abcam), anti-SOX9 (1:200, ab185966; abcam), anti-NKX6.1 (1:200, ab221549; abcam), anti-MAFA (1:200, ab26405; abcam), anti-KI67 (1:200, D3B5; Cell Signaling Technology). Imaging of the expanded islet clusters was performed on Zeiss LSM 780 and processed using ImageJ or Adobe illustrator software.

Lin J.Y., Cheng J., Du Y.Q., Pan W., Zhang Z., Wang J., An J., Yang F., Xu Y.F., Lin H., An W.T., Wang J., Yang Z., Chai R.J., Sha X.Y., Hu H.L., Sun J.P, & Yu X. (2020). In vitro expansion of pancreatic islet clusters facilitated by hormones and chemicals. Cell Discovery, 6, 20.

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Immunohistochemical Staining of Tissues

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Tissues were processed and stained as described (28 (link)) using mouse antiglucagon (1:50; #G2654, Sigma-Aldrich) and guinea pig anti-insulin (1:100, #PU029-UP; Biogenex Laboratories, Fremont, CA) primary antibodies.

Zhang S., Wang S., Puhl M.D., Jiang X., Hyrc K.L., Laciny E., Wallendorf M.J., Pappan K.L., Coyle J.T, & Wice B.M. (2014). Global Biochemical Profiling Identifies β-Hydroxypyruvate as a Potential Mediator of Type 2 Diabetes in Mice and Humans. Diabetes, 64(4), 1383-1394.

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Immunofluorescence Imaging of Pancreatic Cells

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IF was performed as described previously ^{82 (link),83 (link)}. Briefly, after antigen retrieval using citrate buffer (Sigma), 5μm-thick formalin-fixed pancreatic sections were incubated with anti-insulin (1:1000, Agilent Cat#A0564, RRID:AB_10013624) or anti-glucagon primary antibodies (1:1000, Sigma-Aldrich Cat#G2654, RRID:AB_259852) and AlexaFluor-594-conjugated goat anti guinea-pig (1:500, Molecular Probes Cat#A-11076, RRID:AB_141930) and AlexaFluor-488-conjugated goat anti-mouse antibodies (1:500, Thermo Fisher Scientific Cat#A-11001, RRID:AB_2534069). Images were processed for morphometry using ImageJ software by an observer blind to experimental groups.

Duquenne M., Folgueira C., Bourouh C., Millet M., Silva A., Clasadonte J., Imbernon M., Fernandois D., Martinez-Corral I., Kusumakshi S., Caron E., Rasika S., Deliglia E., Jouy N., Oishi A., Mazzone M., Trinquet E., Tavernier J., Kim Y.B., Ory S., Jockers R., Schwaninger M., Boehm U., Nogueiras R., Annicotte J.S., Gasman S., Dam J, & Prévot V. (2021). Leptin brain entry via a tanycytic LepR:EGFR shuttle controls lipid metabolism and pancreas function. Nature metabolism, 3(8), 1071-1090.

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Multicolor Immunostaining of Isolated Islets

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Immunostaining was conducted on isolated islets, fixed in 4% paraformaldehyde before being permeabilised using 0.1% Triton X-100 (Sigma). After blocking with 5% goat serum, islets were incubated overnight (4°C) with primary antibodies before incubation with secondary antibodies. Fluorescent staining was visualised using a laser-scanning confocal microscopy (BioRad) controlled by LaserSharp2000 (BioRad).
Antibodies used in this study were: rabbit FITC 495-conjugated anti-GFP (1:250; DS-PB-00926; InsightBio, Wembley, United Kingdom), mouse anti-glucagon (1:1000; G2654; Sigma), guinea-pig anti-insulin (1:400; A0564; Dako, Santa Clara, California, United States), goat anti-somatostatin (1:100; sc-7819; Santa Cruz Biotechnology, Dallas, Texas, United States); Alexa Fluor 633 goat anti-mouse IgG (1:500; A-21052; ThermoFisher); Alexa Fluor 594 goat anti-guinea pig IgG (1:500; A-11076; ThermoFisher); and Alexa Fluor 546 donkey anti-goat IgG (1:100; A-11056; ThermoFisher).

Acreman S., Ma J., Denwood G., Gao R., Tarasov A., Rorsman P, & Zhang Q. (2024). The endoplasmic reticulum plays a key role in α-cell intracellular Ca2+ dynamics and glucose-regulated glucagon secretion in mouse islets. iScience, 27(5), 109665.

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Multimarker Immunohistochemistry of Pancreatic Islets

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Pancreases or isolated islets from Wistar rats, GK rats and human subjects were fixed in 4% formalin, paraffin embedded and cut into 5‐μm sections. For immunofluorescence, sections were dewaxed and rehydrated as described above. All samples were subjected to antigen retrieval by heating in 0.01 M sodium citrate solution before immunolabelling. Sections were incubated overnight using antibodies targeting PYY (ab22663), insulin (in house), glucagon (G‐2654, Sigma, Gillingham, UK), somatostatin (sc‐25262, Santa Cruz, Insight Biotechnologies, Wembley, UK), PP (ab77192), NPY1R (ab91262, Abcam, Cambridge, UK), NPY2R (ab31894, Abcam), NPY4R (HPA027863, Sigma), NPY5R (ab32886, Abcam) or DPP‐IV (ab28340, Abcam). The tyramide amplification system was used as a secondary antibody system to improve visualization of PYY in human samples, all NPYR proteins and DPP‐IV (ThermoFisher, Loughborough, UK). All other proteins were visualized using fluorescently‐ and HRP‐labelled secondary antibodies for immunolocalization as follows; anti‐rabbit 488 (A11034, Life Technologies, Loughborough, UK), anti‐mouse TRITC (F5262, Sigma), anti‐guinea pig 648 (ab150187, Abcam), anti‐rabbit HRP (P0448, Dako, Cheadle, UK). Images were visualized using a BioRad (Radiance 2100) confocal microscope.

Guida C., McCulloch L.J., Godazgar M., Stephen S.D., Baker C., Basco D., Dong J., Chen D., Clark A, & Ramracheya R.D. (2017). Sitagliptin and Roux‐en‐Y gastric bypass modulate insulin secretion via regulation of intra‐islet PYY. Diabetes, Obesity & Metabolism, 20(3), 571-581.

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Immunofluorescence Analysis of Pancreatic Islets

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Preparation and immunofluorescence of alginate beads were performed as previously described (Vethe et al., 2019b (link)). The pancreas from the mice were fixed for 2 h in 4% PFA, before dehydration using a sucrose gradient of 10, 20, and 30% and embedded in Tissue Tek OCT compound (Sakura JP). Sections of 10 μm were obtained with a cryotome (Leica CM 1950, Leica, DE) and added on SuperFrost Plus slides (Thermo Scientific). The immunofluorescence staining was performed following indications provided by the supplier. The following primary antibodies were used: mouse anti-insulin IgG1 (1/500, I2018, Sigma-Aldrich), guinea-pig anti-porcine insulin (1/400, A056401-2, Dako), mouse anti-porcine glucagon (1/1000, G2654, Sigma-Aldrich). The following secondary antibodies were used at dilution 1/500: goat anti-mouse IgG1 A488, goat anti-guinea-pig A488, goat anti-guinea-pig A546, goat anti-mouse IgG1 A546. DAPI (1/1000, D1306, Molecular Probes) was used to stain the nuclei. The samples were mounted in Prolong Diamond Antifade Mountant Media (P36970, Life technologies). Image acquisition and analysis was performed on Andor Dragonfly confocal microscope and Imaris 9.1.2 (Bitplane AG) as we have previously described (Vethe et al., 2019b (link)). We also performed manual counting on images acquired using a 40x immersion objective on Leica TCS SP5 confocal (Leica Microsystems CMS GmbH).

Legøy T.A., Mathisen A.F., Salim Z., Vethe H., Bjørlykke Y., Abadpour S., Paulo J.A., Scholz H., Ræder H., Ghila L, & Chera S. (2020). In vivo Environment Swiftly Restricts Human Pancreatic Progenitors Toward Mono-Hormonal Identity via a HNF1A/HNF4A Mechanism. Frontiers in Cell and Developmental Biology, 8, 109.

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