Immunofluorescence Staining of Hormones

Immunofluorescence was carried out as previously described [17 (link)] using commercial antisera against glucagon (guinea pig anti-glucagon at 1:7000; EMD Millipore, Billerica MA), somatostatin (sheep anti-somatostatin at 1:1000; American Research Products Inc., Waltham MA), insulin (chicken anti-insulin at 1:1000; Abcam) and IL1r1/CD121a (armenian hamster anti-mouse Il1r1/CD121a-APC at 1:100; Biolegend, San Diego, CA). Rabbit antisera against UCN3 (#7218, 1:2000) and CRH (rc70, 1:5000) were generated in house. Secondary antibodies were raised in donkey against each of these host species and were obtained from Jackson Immuno Research Laboratories (West Grove, PA) conjugated to Alexa Fluor-405, -488, -649 or Cy3 and used at 1:600. Dapi was applied as a nuclear stain at 1 microgram/ml and slides were embedded with Prolong Gold antifade reagent (Life Technologies, Carlsbad, CA). All imaging was carried out on Zeiss LSM780 confocal microscopes at The Waitt Advanced Biophotonics Center Core facility of the Salk Institute.

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Benner C., van der Meulen T., Cacéres E., Tigyi K., Donaldson C.J, & Huising M.O. (2014). The transcriptional landscape of mouse beta cells compared to human beta cells reveals notable species differences in long non-coding RNA and protein-coding gene expression. BMC Genomics, 15(1), 620.

Publication 2014

guinea pig anti-glucagon chicken anti-insulin Prolong Gold antifade reagent

Antibodies Antisera Armenian hamster Cd121a Chicken Confocal microscopes Dapi Donkey Glucagon Gold Guinea pig Host species Immunofluorescence Insulin Mouse Rabbit Sheep Somatostatin Stain Ucn3

Corresponding Organization :

Other organizations : Salk Institute for Biological Studies

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Variable analysis

independent variables

Antisera against glucagon (guinea pig anti-glucagon at 1:7000; EMD Millipore, Billerica MA)
Antisera against somatostatin (sheep anti-somatostatin at 1:1000; American Research Products Inc., Waltham MA)
Antisera against insulin (chicken anti-insulin at 1:1000; Abcam)
Antisera against IL1r1/CD121a (armenian hamster anti-mouse Il1r1/CD121a-APC at 1:100; Biolegend, San Diego, CA)
Antisera against UCN3 (#7218, 1:2000)
Antisera against CRH (rc70, 1:5000)

dependent variables

Immunofluorescence staining patterns

control variables

Dapi as a nuclear stain at 1 microgram/ml
Prolong Gold antifade reagent (Life Technologies, Carlsbad, CA) for embedding slides
Imaging carried out on Zeiss LSM780 confocal microscopes at The Waitt Advanced Biophotonics Center Core facility of the Salk Institute

controls

Positive controls: Not specified
Negative controls: Not specified

Annotations

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