Alexa fluor 488 goat anti guinea pig

Immunofluorescence staining was performed as described previously.^{29 (link)} Hippocampal cells were fixed in PBS containing
4% PFA for 20 min at RT. Cells were permeabilized with 1% Triton X-100
for 30 min, blocked with 5% FBS in PBS for 30 min at room temperature
(RT), and incubated with primary antibodies for 60 min. The primary
antibodies used were mouse polyclonal anti-β-tubulin III (Sigma,
1:250 dilution), rabbit polyclonal anti-NPY1R (Abcam Plc, 1:500 dilution),
and rabbit polyclonal anti-NPY2R (Thermo Fisher, dilution 1:2000).
After the primary incubation and PBS washes, neurons were incubated
for 60 min with the secondary antibodies Alexa Fluor 488 goat anti-rabbit
(Invitrogen, dilution 1:500), Alexa Fluor 594 goat anti-mouse (Invitrogen,
dilution 1:500), Alexa Fluor 488 goat anti-guinea pig (Invitrogen,
dilution 1:500), and DAPI (Invitrogen, dilution 1:200) to stain the
nuclei. Samples were mounted in VECTASHIELD (Vector Laboratories)
on 1 mm thick coverslips. Image acquisition was performed using a
confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) with
63× (1.4 NA) magnification (Z-stacks were acquired
every 150 nm).

Brains of 3^rd instar larvae were dissected in 0.7% NaCl, incubated for 10 min in 0.5% Trisodium citrate and fixed 8 min in 3.7% formaldehide + 45% acetic acid on a cover-slip pre-treated with Silanization Solution II (Fluka). Brains were squashed and washed in 0.1% PBT (PBS/0.1% Triton X-100) and blocked in PBTM (PBT/1% non-fat dry milk) for 1 hr. Samples were incubated with α-HeT-A Gag (guinea pig; obtained from Dr. Yikang Rong) 1:1000 and α-TART Pol (rabbit) 1:50 primary antibodies diluted in PBTM overnight in a humid chamber at 4°C. A wash with PBT was followed by 3, 15 min washes with PBTM. Slides were incubated with secondary antibodies [Alexa Fluor 488 goat anti-guinea pig (Invitrogen), Alexa Fluor 555 goat anti-rabbit (Invitrogen)] in PBTM 1 hr at room temperature in the dark and washed with PBT and PBS. A drop of DAPI-containing Mowiol medium was added to the slide and covered with a coverslip. Images were obtained using the Zeiss Axio Imager.Z2 fluorescence microscope using 20x, 40x and 63x objectives.

Rabbit polyclonal IgG antibodies against Su(Hw), Mod(mdg4)67.2, and CP190 and rat polyclonal IgG antibody against Su(Hw) were previously generated by our lab (Wallace et al, 2010 (link); Schoborg et al, 2013 (link)). Mouse monoclonal antibodies against the phosphorylated form of H2Av (Lake et al, 2013 (link)) were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa, Department of Biology, Iowa City, IA 52242. Polyclonal rabbit antibodies against H2Av were purchased from Active Motif (RRID:AB2793318). The monoclonal Mouse antibody anti-myc was used to detect Rad21::myc and was obtained from Ubiquitin-Proteasome Biotechnologies (#Y1090; UBPBio). All primary and secondary antibodies were diluted 1:1 in glycerol (BP229-1, lot 020133; Thermo Fisher Scientific) and used at a final dilution of 1:200. The following secondary antibodies were used in this study: Alexa Fluor 594 goat anti-rabbit (A-111037, lot 2079421; Invitrogen), Alexa Fluor 488 donkey anti-rabbit (lot 1834802, A-21206; Invitrogen), Alexa Fluor 488 goat anti-guinea pig (lot 84E1-1, A-11073; Invitrogen), Texas red donkey anti-rat (712-075-150; Jackson Immuno-Research Laboratories), and Alexa Fluor 488 goat anti-mouse (lot 1858182, A-11001; Invitrogen).

Alexa Fluor secondary antibodies (Invitrogen) were used at a dilution of 1:1,000.
Alexa Fluor 488 goat anti-mouse (#A11029), Alexa Fluor 488 goat anti-rabbit (#A11034), Alexa Fluor 488 goat anti-guinea pig (#A11073), Alexa Fluor 488 goat anti-chicken IgY (#A11039), Alexa Fluor 555 goat anti-mouse (#A21422), Alexa Fluor 555 goat anti-rabbit (#A21428), Alexa Fluor 568 goat anti-guinea pig (#A11075), Alexa Fluor 647 goat anti-mouse (#A21236), Alexa Fluor 647 goat anti-rabbit (#A21245). Only cross-adsorbed secondary antibodies were used in this study to eliminate the risk of cross-reactivity.
F-Actin was stained with phalloidin conjugated to Rhodamine (Invitrogen, Cat. #R415, 1:500 dilution).