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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.

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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.

Publication 2019

A0564 GW10064F G2654 A0565 A0566 sc-10368 ab-13970 ab28364 ab92547 ab152 A0010 Alexa 405 FITC Cy3 TRITC TCS SPE

Anti synaptophysin Antibodies Chicken Confocal microscope Dapi Fitc Frozen sections Ghrelin Glucagon Goat Guinea pig Immunofluorescence Insulin Mouse Pancreatic polypeptide Pdx1 Porcine insulin Rabbit Scan Somatostatin Tritc Tyrosine hydroxylase Vimentin

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Variable analysis

independent variables

Primary antibodies used

dependent variables

Immunofluorescence signals of the proteins detected by the primary antibodies

control variables

Frozen sections were cut at 10 μm-thick
Sections were counterstained with DAPI
All sections were examined with a confocal microscope (Leica TCS SPE)

controls

Positive controls: No positive controls were explicitly mentioned.
Negative controls: No negative controls were explicitly mentioned.

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