Alexa 568 conjugated goat anti rabbit

Neurons grown on glass coverslips were fixed with 4% (vol/vol, in PBS) paraformaldehyde for 30 min and immunostained with anti-PCNA (1:100; 24; 610664, BD Transduction Laboratories). Immunolabeling was deteted by using Alexa 568-conjugated goat anti-mouse (1:500) (Jackson ImmunoResearch) or Alexa 568-conjugated goat anti-rabbit (1:500) (Molecular Probes, Invitrogen). Coverslips were washed, mounted in SlowFade light antifade reagent (Invitrogen) on glass slides, and examined using a microscope (Provis AX70, Olympus) equipped with epifluorescence and appropriated filters sets.

Fly heads were dissected in HL3 hemolymph-like solution, fixed for four hours in ice-cold 4% para-formaldehyde in filtered PBS, washed overnight in 25% (wt/vol) sucrose in phosphate buffer (pH 7.4), embedded in Optimal Cutting Temperature compound (EMS, Hatfield, PA), frozen in dry ice and sectioned at 20 μm thickness on a cryostat microtome (CM 1950, Leica Microsystems, Wetzlar, Germany). Sections were probed overnight with primary antibodies against Drosophila BiP (1:2000, Gift from Don Ryoo (Ryoo et al., 2007 (link)), GFP (1:1000, A10262, ThermoFisher Scientific, Waltham, MA) or RFP (1:1000, 600-401-379, Rockland, Limerick, PA). Secondary antibodies were labeled with Alexa488-conjugated Goat anti-Chicken (Molecular Probes, P/N# A-11039), Alexa488-conjugated Goat anti-Guinea Pig (Molecular Probes, P/N# A-11073), or Alexa568-conjugated Goat anti-Rabbit (Molecular Probes, P/N# A-11011). Alexa 647-conjugated phallodin was also added to label Actin for identifying structures. Images were captured with an oil-immersion 63 × NA−1.4 lens on an inverted confocal microscope (LSM710, Carl Zeiss). For each genotype and light rearing conditions, immunohistochemistry experiments were performed in two biological replicas with new sets of flies, using identical acquisition settings. Blinding of the samples to the user acquiring the images was performed when appropriate.

Whole-mount immunohistochemistry staining was performed as previously described^{121 (link)}. Animals were sacrificed with 2% HCl and fixed with 4% FA. Animals were blocked in 1% bovine serum albumin (BSA) in 1X PBSTx 0,3% (Blocking Solution) for 2 h at RT. Primary antibodies were diluted in blocking solution and incubated 16 h rocking at 4 °C. Then, washes were per performed for at least 4 h. Secondary antibodies were diluted in blocking solution for 16 h rocking at 4 °C.
The following antibodies were used in these experiments: mouse anti-synapsin (anti-SYNORF1, 1:50; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), rabbit anti-phospho-histone H3 (Ser10) (D2C8) (PH3) (1:500; Cell Signaling Technology) and mouse anti-VC1 (anti-arrestin, 1:15,000, kindly provided by Professor K. Watanabe). The secondary antibody used was Alexa 488-conjugated goat anti-mouse (1:400; Molecular Probes, Waltham, MA, USA) and Alexa 568-conjugated goat anti-rabbit (1:1000: Molecular Probes, Waltham, MA, USA). Nuclei were stained with DAPI (1:5000; Sigma).