Eclipse ti a1

Cryo-sections were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 2% bovine serum albumin and incubated with Anti-Cardiac Troponin I at 4 °C overnight. The next day, the TUNEL assay was performed using the TUNEL BrightRed Apoptosis Detection Kit (Vazyme, China) according to the manufacturer’s instructions. DAPI was used for nuclear staining, and the number of TUNEL-positive nuclei was counted. All sections were examined by confocal microscope (ECLIPSE Ti A1, Nikon).

The distribution of FITC-labeled insulin in the SFD particles was evaluated using a Nikon Eclipse Ti A1 Laser Scanning Confocal Imaging System (Nikon, Tokyo, Japan) equipped with a modular laser system and an inverted Nikon microscope. Insulin was labeled with fluorescein isothiocyanate (FITC) based on the reaction between the isothiocyanate group of FITC and the amine groups of insulin, as previously described [18 (link),19 (link)]. Then, 5 mg of FITC was dissolved in 1 mL of DMSO and added drop by drop into a 20 mL of 0.1 M sodium carbonate solution containing 100 mg of insulin. Upon addition of FITC, the reaction vials were protected from light and mixed under magnetic stirring for 12 h at 4 °C. 7 mL of 0.2 M ammonium chloride solution was added into the reaction vials under magnetic stirring for 2 h to quench the excess FITC. The mixture was then dialyzed (1000 MWCO) and lyophilized to obtain the FITC-labeled insulin powder. SFD particles were prepared with labeled insulin to determine the distribution of insulin in the SFD particles under confocal microscope with the argon-ion-laser line (excitation: 488 nm/Emission: 524 nm).

All confocal imaging was performed using the Purdue College of Pharmacy Live Cell Imaging Facility Nikon Eclipse Ti A1 instrument using NIS-elements AR software to capture 1024 × 1024 pixel resolution images at 1/4 frame/second on 60× oil objectives detecting the fluorophores with channels in series.