Imaging GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Begin with wild-type and CCR5-knockout mouse primary hypothalamic neurons cultured on poly-D-lysine-coated coverslips in a multi-well plate.

These neurons express green fluorescent protein-tagged glucose transporter-4 (GFP-GLUT4) in the cytoplasm.

Transfer the coverslips onto glass slides and gently wipe off excess medium to ensure stability during image acquisition.

Place a slide on the deconvolution microscope stage with the coverslip facing downward.

Visualize the neurons under bright-field illumination, then treat them with the chemokine CCL5 and insulin sequentially.

Switch to fluorescence illumination to identify GFP-GLUT4-expressing target neurons and adjust the imaging parameters to capture live fluorescent images.

In wild-type neurons, insulin and CCL5 bind to the insulin receptor and CCR5, respectively, activating downstream signaling pathways that drive GFP-GLUT4 membrane translocation.

In CCR5-knockout neurons, CCL5 fails to bind, resulting in reduced GFP-GLUT4 translocation.

Capture the images and apply a deconvolution algorithm to reduce out-of-focus light, enhancing image clarity and resolution.

To prepare the cells for live imaging, carefully remove the coverslips with neu