Dylight 594 anti mouse

Immunofluorescent staining of L6-GLUT4myc myoblasts was performed as previously described [22 (link)] to determine the colocalization of EXOC5 and CD36 in response to insulin and ionomycin treatment.
L6-GLUT4myc myoblasts were grown on coverslips to 70–80% confluence. Following treatment with insulin or ionomycin, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized. After blocking with 0.1% BSA in PBS, samples were incubated overnight in primary antibodies anti-EXOC5 (Cat# sc-514802, Santa Cruz Biotechnologies, Dallas, TX, USA) and anti-CD36 (Cat# NB400-144, Novus Biologicals LLC, Centennial, CO, USA) at 4 °C. The samples were subsequently incubated with secondary antibodies (DyLight 488 Anti-Rabbit IgG and DyLight 594 Anti-Mouse, Vector Laboratories, Newark, CA, USA) at a 1:1000 dilution at room temperature for 1 h. We used DAPI as a nuclear stain and mounted the samples with Fluoromount-G mounting medium (Thermo Fisher Scientific, Waltham, MA, USA). Imaging was performed using a Leica SP8 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany).

All samples were imaged using an Olympus BX41 microscope. Image processing, quantification of pancreatic islet areas, and oil red staining were performed using ImageJ (39 ).
For immunofluorescence, deparaffinized and rehydrated sections were subjected to a citrate-based antigen retrieval. After permeabilization and blocking, sections were incubated with primary antibodies (anti-insulin (Cat #66198-1-Ig, Proteintech Group, Inc); anti-glucagon (Cat # 15954-1-AP,Proteintech Group, Inc)) overnight at 4 °C and incubated with appropriate fluorescent secondary antibodies (DyLight 488 Anti-Rabbit IgG and DyLight 594 Anti-Mouse, Vector Laboratories) for detection. Cell nuclei were stained with DAPI.
In the sections stained for insulin and glucagon, the borders of each pancreatic islet were identified morphologically and marked using the pen tool in ImageJ. The area of each islet was calculated with ImageJ software using the set scale function corresponding to the 20x scale bar. Insulin-positive nuclei were counted based on fluorescent signal. All islets with an area >566 μmˆ2 (corresponding to a diameter of >27 μm), approximately corresponding to islets containing a minimum of eight nuclei, were regarded for analysis.