Goat anti mouse alex 488

Larval brains collected at 120 h AEL were dissected and fixed in 4% PFA/PBS at room temperature for 30 min, followed by washing in PBST (0.3% Triton-X 100 in PBS), and incubating in the primary antibody overnight at 4 °C. On the next day, brains were washed with PBST and incubated in the secondary antibody at room temperature for 1 h before final washes in PBST and mounted on the slide with the anti-fade mounting solution. Primary antibodies used were mouse anti-PDF (DSHB PDF C7, 1:10), mouse anti-Chp (DSHB 24B10, 1:10) and rat anti-HA (Sigma, 11867423001, 1:200). Secondary antibodies (1:1000 dilution) used were goat anti-mouse Alex 488 (Invitrogen, A-32723), goat anti-mouse Alex 647 (Invitrogen, A-32728), donkey anti-mouse CY3 (Jackson Immuno Research Labs, 715165150), goat anti-rat Alex 647 (Invitrogen, A-21247). Images are taken either with either a Zeiss LSM700 confocal microscope in the lab or Zeiss LSM800 confocal microscope at NINDS Neurosciences Light Imaging Facility.

The WT, ΔSAA, KSEH truncation (ΔKSEH), and cholesterol modification site mutation C203* plasmids were transfected into ECHO cells as described above. Next, 24 h after ponasterone A was added, anti-FLAG monoclonal antibody was incubated at 37 °C for 2 h to stain the IHHNp on the cell membrane surface. Then, cells were fixed with 80% cold acetone for 30 min at 4 °C and labelled with goat anti-mouse Alex488 (Invitrogen, A32723, 1:1000) for 2 h at 37 °C. A Leica SP8 laser scanning microscope was used for detecting immunofluorescence.