In all recordings, cell identity (α, β or δ) was subsequently established by immunocytochemistry. Biocytin (0.5 mg ml−1) was included in the intracellular solution to allow identification of the cell recorded from. Following voltage-clamp experiments, islets were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) overnight and permeabilized with 0.3% Triton X-100. Non-specific binding was blocked by pre-treatment for 2 h with 5% normal goat serum before incubating with the different primary antibodies for 4–12 h (guinea pig anti-insulin (Abcam, Cambridge, UK), sheep anti-glucagon (Sigma-Aldrich, St Louis, MO) and rabbit anti-somatostatin (Vector Labs, Burlingame, CA)). After washing with PBS, the islet was incubated for 1 h in secondary antibodies (Alexa 633 goat anti-guinea pig (insulin), Alexa 405 goat anti-mouse (glucagon) and Alexa 543 goat anti-rabbit (somatostatin)). Biocytin labelling was visualized by using Alexa Fluor 488 conjugated streptavidin (0.04 mg ml−1; Thermo Fisher). Islets were then washed and imaged on a confocal microscope (Axioskop 2 upright microscope fitted with a Zeiss LSM 510 meta confocal and a chameleon multiphoton module).
Axioskop 2 upright microscope
Manufactured by Zeiss
Sourced in United States
The Axioskop 2 is an upright microscope designed for a range of applications in biological and materials science research. It features high-quality optics and a modular design that allows for customization to meet specific user requirements. The core function of the Axioskop 2 is to provide a stable and reliable platform for high-resolution imaging and analysis of samples.
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Lab products found in correlation
Alexa fluor 488 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Lsm 510 meta confocal, by Zeiss (1 mentions)
Guinea pig anti insulin, by Abcam (1 mentions)
Sheep anti glucagon, by Merck Group (1 mentions)
Rabbit anti somatostatin, by Vector Laboratories (1 mentions)
Lsm 5 exciter laser scanning confocal system, by Zeiss (1 mentions)
Hypromellose, by Akorn (1 mentions)
2 protocols using axioskop 2 upright microscope
1
Immunocytochemical Identification of Islet Cells
2
In vivo Fluorescence Imaging of Retinal Ganglion Cells
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The Thy1-CFP mice were anesthetized with isoflurane (2%; MWI Veterinary Supply, Inc., Meridian, ID, USA) in 95% O2 and 5% CO2, and placed on a temperature-controlled heated platform at 38°C. Topical tropicamide and 1% atropine sulfate ophthalmic solution (both Bausch & Lomb Pharmaceuticals) were used to dilate the pupils. A drop of hypromellose (2.5%; Akorn Pharmaceuticals, Lake Forest, IL, USA) was placed in the eye and a glass coverslip was placed over it to create a planoconcave lens on the mouse cornea for in vivo imaging. An LSM 5 Exciter Laser scanning confocal system (Carl Zeiss Inc., Thornwood, NY, USA) was modified (using an Axioskop 2 upright microscope equipped with a LSM 5 Exciter Laser scanning confocal system) to visualize CFP [blue-light cSLO (bCSLO); 460 nm excitation and 490 nm detection]. The number of CFP+ RGCs in the same area of the retina were assessed in vivo once a week for 6 weeks.
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