C2 scanning confocal microscope

Cerebella and lumbar spinal cord tissues were isolated during EAE onset, immunostained with IBA1 and scanned using a 60x oil objective on the Nikon C2 scanning confocal microscope. The simple neurite tracing tool on Fiji ImageJ software (NIH) was used to trace IBA1 + branches. Two images per section, 4–8 microglia per section, two sections per tissue and 6–7 mice were employed.

First, 2.5 × 10⁴ cells/well were seeded in 8-well μ-slides (Ibidi^®, #80826) and incubated at 37°C for 12 h. The cells were treated with vehicle, compounds Ganoderic acid A or EP, or cycloheximide (positive control) for 2 h to determine protein synthesis as nascent proteins generated using Biovision’s EZClick^TM Global Protein Synthesis Assay Kit, (#K715-100, Milpitas, CA, United States). The assay includes a robust chemical method based on an alkyne containing o-propargyl-puromycin (OP-puro) probe, which stops translation by forming covalent conjugates with nascent polypeptide chains. Truncated polypeptides are rapidly turned over by the proteasome and can be detected based on the subsequent click reaction with a fluorescent azide. The reaction was conducted according to the manufacturer’s protocol. Cells were imaged at 20× with a Nikon C2 scanning confocal microscope, and the whole image montage was quantified by using Gen5 Software and Lionheart^TM FX Automated Microscope (BioTek, Winooski, VT, United States).