For generation of anti-Nidogen antibody, rabbits were immunized with epitope CTYVQEFDGERNADLIPC by Bio-med Biotechnology (Beijing, China). Embryos, fat bodies, wing imaginal discs and ovaries were stained using standard procedures and mounted in DAPI-Vectashield (Vector Laboratories, Burlingame, California). The following primary antibodies were used: rabbit anti-Ndg (1:2000, this study), chicken anti-betagalactosidase (1:500, AbCam, Cambridge, UK), chicken anti-GFP (1:500, AbCam), rabbit anti-Ndg (1:100, [34 (link)]). Secondary antibody is IgG conjugated to Alexa-555, IgG conjugated to Alexa-488 and Alexa 549 (1:200, Life technologies).
For lipid droplet staining, L3 larvae were turned inside out and fixed in 4% PFA for 20 minutes, washed twice in PBS and then incubated in a 1:1000 dilution in PBS of 1 mg/ml BODIPY 493/503 stock (Life Technologies) for 30 minutes, followed by two 10-min washes in PBS and mounting in DAPI-Vectashield (Vector Laboratories). Confocal images were obtained using a Leica (Wetzlar, Germany) SP2 microscope or a Zeiss (Oberkochen, Germany) LSM780 microscope equipped with a Plan-Apochromat 63X oil objective (NA 1.4). Eggs and pupae were imaged in a Leica M125 stereoscope. All images were processed with Adobe Photoshop and ImageJ.
For lipid droplet staining, L3 larvae were turned inside out and fixed in 4% PFA for 20 minutes, washed twice in PBS and then incubated in a 1:1000 dilution in PBS of 1 mg/ml BODIPY 493/503 stock (Life Technologies) for 30 minutes, followed by two 10-min washes in PBS and mounting in DAPI-Vectashield (Vector Laboratories). Confocal images were obtained using a Leica (Wetzlar, Germany) SP2 microscope or a Zeiss (Oberkochen, Germany) LSM780 microscope equipped with a Plan-Apochromat 63X oil objective (NA 1.4). Eggs and pupae were imaged in a Leica M125 stereoscope. All images were processed with Adobe Photoshop and ImageJ.