Lsm 710 meta confocal microscope

Embryos were fixed in 4% formaldehyde, phosphate buffer pH 7.4 as previously described [45 (link)]. The primary antibodies used for indirect immunofluorescence were as follows: guinea pig anti PATJ (1 : 500, [30 (link)]), mouse anti Sdt (1 : 20, [42 (link)]), rabbit anti Sdt (1 : 2000, this study), rabbit anti Baz (1 : 1000, [43 (link)]), mouse anti Crb (Cq4, 1 : 50, DSHB), mouse anti Dlg (1 : 50, DSHB), rabbit anti GFP (#A11122, 1 : 1000, Life Technologies) and chicken anti GFP (1 : 2000, Aves Laboratories). Secondary antibodies conjugated with Alexa 488, Alexa 568 and Alexa 647 (Life Technologies) were used at 1 : 400.
Images were taken on a Zeiss LSM 710 Meta confocal microscope and processed using Adobe Photoshop.

Immunocytochemistry of cells was carried out as described previously.^{2 (link)} The paraffin-embedded tumor samples were de-paraffinized and afterwards rehydrated with in Xylol, followed by a decreasing alcohol series (100% to 70% alcohol). The rehydration was completed by incubation with bidest H₂O and slices were stored in water until antigen detection. Afterwards immunostaining was performed as described previously.^{2 (link)} Cells and tissue were analyzed by microscopy using an inverted Leica TCS SP5 (Wetzlar, Germany) and Zeiss LSM710 meta confocal microscope (Oberkochen, Germany) and images were processed with Adobe Photoshop CS5 (San Jose, CA, USA) and Leica LAS AF lite software (Leica Microsystems (UK) Ltd, Milton Keynes, UK). GFP-RFP-LC3 dot formation (visualized using the GFP-RFP-LC3 expression plasmid mentioned above) was quantified by counting dots in confocal laser scanning microscopic pictures of corresponding transfected cell lines and after Bafilomycin A₁ treatment (500 nM, 6 h). At least 56 cells per cell line were analyzed.