Uorophore conjugated secondary antibodies

Immunostaining of the larval samples was performed as described previously ( [44] ). Brie y, the wandering larval samples were dissected in Jan solution; xed in paraformaldehyde at room temperature;
washed with PBS and 0.3% PBST (0.3% Triton X-100 in PBS); blocked in 1% BSA for 1 hour; incubated with anti-Hrp (Jackson ImmunoResearch, West Grove, PA), anti-synaptotagmin (1:50), and anti-synapsin (1:50) antibodies at 4°C for 2 hours; and incubated with uorophore-conjugated secondary antibodies (Invitrogen, 1:500) for 1 hour at room temperature. The samples were washed extensively with PBST and mounted in VectaShield mounting medium (Vector Laboratories). Images were collected using an Olympus FV3000 confocal microscope. During observation of degenerated NMJ boutons, the large pinhole of the confocal microscope was adjusted to increase the thickness of a single optical section, and the 3D analysis function of the confocal microscope was utilized to observe the complete NMJ bouton in the 6th/7th muscles in the A 3 or A 2 segment.

The RA-FLS were xed with 4% paraformaldehyde at 48 h post transfection, washed three times with PBS and blocked with 2% BSA in PBS for 15 min. After blocking, cells were incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. The RA-FLS were washed three times with PBS and incubated for 1 h with uorophore-conjugated secondary antibodies (1:1000; Invitrogen) in blocking buffer. Cell nuclei were stained with DAPI (Invitrogen). Images were acquired using a Zeiss LSM710 confocal microscope.