Lcs version 2

Semi-thin sections were blocked by 5% (w/v) BSA and incubated for 1 h with the corresponding primary polyclonal antibody diluted in 1% BSA at 1:100 (HvPap-1), 1:50 (HvPap-6), 1:20 (HvPap-19), and 1:50 (ATG5 and ATG8). After washing in PBS, signal was revealed with Alexa Fluor 488-labelled anti-rabbit IgG antibody (Molecular Probes) diluted 1:25 in 1% BSA for 45 min in darkness. Finally, sections were counterstained with 1 mg ml⁻¹ DAPI for 10 min and analysed by confocal laser microscopy (Leica TCS-SP5-AOBS, Vienna, Austria). Images of maximum projections were obtained with software of the confocal microscope (Leica software LCS version 2.5). Negative controls were performed avoiding the primary antibody.

Semithin sections of immature zygotic embryos, PEMs, and heart and torpedo embryos were blocked by 10% (w/v) fetal calf serum (FCS) in PBS for 10 min, washed in 1% PBS, and incubated for 1 h with anti-IAA mouse monoclonal antibody (Sigma, cat. no. A0855) which had been diluted 1:100 in 1% (w/v) bovine serum albumin (BSA) in PBS. After washing in 1% PBS, the signal was revealed with Alexa Fluor 488-labeled anti-mouse IgG antibody (Molecular Probes, Eugene, OR, USA) which had been diluted 1:25 in 1% (w/v) BSA in PBS for 45 min in darkness. Afterwards, sections were washed in 1% PBS, counterstained with 1 mg/mL 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) for 10 min, and washed again in 1% PBS. Finally, sections were mounted in Mowiol and analyzed using a confocal laser microscope (Leica TCS-SP5-AOBS, Vienna, Austria). Maximum projection images were obtained using confocal microscopy software (Leica software LCS version 2.5). The confocal microscopy analysis was performed using the same laser excitation and sample emission capture settings for image acquisition for all immunofluorescence preparations, allowing an accurate comparison to be made among signal intensities. Controls were created by omitting the primary antibody in the immunofluorescence assay.