Rabbit phospho ampk

Cells were cultured for 2 days and fixed with 4% paraformaldehyde (PFA) for 10 min, permeabilised in 0.4% TX-100 in phosphate-buffered saline (PBS) for 10 min, blocked in 5% normal goat serum in PBS for 30 min and incubated with rabbit phospho-AMPK (1∶100, Cell Signaling Technology) antibody in blocking buffer overnight at 4°C, washed three-times with PBS, for 10 min and incubated with Alexa Fluor 488 conjugated secondary antibody (1∶1,000, Molecular Probes) for 2 hr, stained with Hoechst 33342 (1∶10,000, Invitrogen) for 15 min, washed three times with PBS for 10 min and mounted using fluorescent mounting medium (Dako) on glass slides (Thermo Scientific) for microscopy using an Olympus FV 1000 confocal microscope. Images were captured using identical exposure and gain settings. Negative controls without primary antibodies were performed which produced no staining.

Mice were transcardially perfused with PBS followed by 4% (w/v) PFA in 0.1 M phosphate buffer. Lumbar spinal cords were dissected out, post-fixed in 4% (w/v) PFA for 2 hr, processed, dehydrated and embedded in paraffin and cut into 20 μm transverse sections. Sections were deparaffinised, treated with 20 μg/ml Proteinase K (Qiagen) in PBS at 37°C for 5 min for antigen retrieval, permeabilised in 0.4% TX-100 in PBS for 10 min, blocked in 5% normal goat serum in PBS for 30 min and incubated with rabbit phospho-AMPK (1∶100, Cell Signaling Technology) and mouse NeuN (1∶1,000, Millipore, MAB377) antibodies in blocking buffer overnight at 4°C. Sections were washed three times with PBS for 10 min, incubated with Alexa Fluor 488 and Alexa Fluor 594 conjugated secondary antibodies (1∶1,000, Molecular Probes) for 2 h, stained with Hoechst 33342 (1∶10,000, Invitrogen) for 15 min, and washed three times with PBS for 10 min before mounting using fluorescent mounting medium onto glass slides for confocal microscopy. The number of cells with pAMPK granules was counted from 30 motor neurons per mouse, 3 mice per group, and expressed as a percentage of WT (100%). The number of pAMPK granules per cell was counted from 30 motor neurons per mouse, 3 mice per group.