Alexa fluor 546 anti rat igg

For immunostaining of the adult brain, the brains of 2- to 7-day-old flies of the relevant genotypes were dissected in PBS and fixed in 4% paraformaldehyde in PBS for 60 min on ice. After washing in PBT (PBS with 0.5% Triton X-100), the tissues were kept in PBTN (PBT with 10% normal goat serum) for 60 min at room temperature (rt) for blocking, and reacted in PBTN for 24 h at 4 °C with the following primary antibodies and dilutions: rabbit anti-GFP at 1:500 (Invitrogen, A6455); mouse nc82 at 1:10 (Developmental Studies Hybridoma Bank); guinea pig anti-FruMale at 1:500^{29 (link)}; chicken anti-GFP at 1:500 (abcam, ab13970); rabbit anti-Tei at 1:100 (this study), and rat anti-HA at 1/500 (Roche, 11867423001). After 1 h of washing in PBT, the tissues were incubated for 24 h at 4 °C in the following secondary antibodies and dilutions: Alexa Fluor488 anti-rabbit IgG; Alexa Fluor546 anti-mouse IgG; Alexa Fluor546 anti-guinea pig IgG; and Alexa Fluor546 anti-rat IgG (all at 1/200 and all from Invitrogen). Samples were washed for 1 h in PBT before being mounted with VECTASHIELD mounting medium (Vector Laboratories Inc.). Stacks of optical sections at 1 μm were obtained with a Zeiss LSM 510 META confocal microscope and processed with the software packages NIH Image J (http://rsb.info.nih.gov/ij/) and Adobe Photoshop.

Retina sections were deparaffinated in Bond Dewax Solution (Leica Biosystems Inc., Buffalo Grove, IL, USA) and rehydrated in alcohol solutions. To retrieve antigens, Epitope Retrieval Solution (Leica Biosystems Inc., Buffalo Grove, IL, USA) was used at 60 °C overnight, before slides were blocked in a solution of phosphate-buffered saline (PBS) with 2% bovine serum albumin (BSA). Then, slides were incubated for 2 hrs at room temperature with rat monoclonal Iba1 (ab-283346; Abcam PLC., Cambridge, UK; 1:100) and rabbit polyclonal LC3B (ab48394; Abcam; PLC., Cambridge, UK; 1:200) antibodies, both diluted in primary antibody diluting buffer (Bio-Optica, Milano, Italy). After the PBS washing steps, AlexaFluor 488 anti-rabbit IgG (a-11034; Invitrogen, Waltham, MA, USA) and AlexaFluor 546 anti-rat IgG (a-11081; Invitrogen, Waltham, MA, USA) secondary antibodies, both diluted in PBS 1:400, were applied in the dark for 30 min at room temperature. Slides were then washed again with PBS to counterstain cell nuclei with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St Louis, MO, USA) and mounted the stained slides with CC/Mount aqueous mounting medium (Sigma-Aldrich, St Louis, MO, USA), before their examination with a Leica TSC SP8 laser scanning confocal microscope [73 (link)].