Lsm 780

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.

Following immunostaining, the brain sections were imaged using a spinning disk microscope and labeled NPC cells were counted using optical dissector stereologic technique [41] (link). All fluorescence immunostaining images were collected employing a Perkin-Elmer UltraVIEW VoX high speed spinning disk (Yokogawa CSU-X1) laser confocal microscope and Volocity software. All representative images were taken by Zeiss LSM710 and LSM780 and imported into Adobe Photoshop Version 12.0 (Adobe Systems Incorporated, San Jose, CA). Brightness, contrast, and background were adjusted using the “brightness and contrast” controls.