Alexa fluor 555 donkey anti rabbit

Immunofluorescence staining was performed according to standard protocols using the following antibodies: Alexa Fluor 555 donkey anti-rabbit (1:100; Life Technologies, Eugene, OR, USA), Alexa Fluor 488 donkey anti mouse (1:100; Life Technologies, Eugene, OR, USA), cy5 donkey anti goat (1:100; Invitrogene, Eugene, Oregon, USA), OCT3/4 (1:100; Millipore, Santa Cruz, CA, USA), SSEA4 (1:100; Millipore, Temecula, CA, USA), TRA1-60 (1:100; Millipore, Temecula, CA, California), Nanog (1:50; R&D, Minneapolis, MN, USA), DAPI (1:500; Sigma Aldrich, St. Louis, MO, USA).

The animals were perfused transcardially with a phosphate-buffered (0.1 M; pH 7.4) paraformaldehyde solution (4%). The brain was removed and postfixed for 3 h, then cryoprotected with 30% sucrose in PBS, and cut at 30 μm on a freezing microtome. The immunohistochemical procedures were performed according to standardized protocols [8 (link)]. Primary antibodies were polyclonal rabbit anti-Cox-2 (Santa Cruz, M-19, sc-1747-R; diluted 1:500), detected with Alexa Fluor 555 donkey anti-rabbit, 1:1000 (Life Technologies, Cat. no. A31572), and purified rat monoclonal anti-CD31 (AbD Serotec, MCA2388GA; 1:1000), detected with Alexa Fluor 488 donkey anti-rat (Life Technologies, Cat. no. A21208; 1:500).

A 1% gelatin (Sigma-Aldrich) pre-coated 12 mm diameter coverslip (Epredia™ Menzel™, Fisher Scientific) was placed in each well of a 24-well plate and 10⁵ cells added and cultured until 70–80% confluency. Cells were then fixed with 4% paraformaldehyde for 15 min at room temperature after which wells were rinsed three times with PBS prior to addition of 500 μl permeabilization solution (PBS + 0.25% Triton X-100). After washing with PBS a 2.5% BSA solution was applied for 1 h and then incubated with primary anti-human XOR rabbit antibody (1:25, Abcam 133,268 RRID: AB_11154903) and left over-night at 4 °C. The following day coverslips were washed with PBS and incubated with the secondary antibody, Alexa Fluor™ 555 donkey anti-rabbit (1:250, Life Technologies, RRID: AB_10892947) for 1 h at room temperature. Each coverslip was then washed prior to incubation with PBS + DAPI (1:5000) staining solution. For each mutant two coverslips were analysed for each experiment with XOR expression visualized under a confocal microscope (LSM 880 with Airyrscan equipped with an Apochromat 63 × ; 1.4 numerical aperture (NA) oil objective (Zeiss, Germany)).

Mice were sacrificed and perfused with 4% PFA in PBS. Brains were fixed overnight in PFA, cryoprotected in 30% sucrose (wt per vol) at 4 °C for 24 h, and then coronally sectioned at 40 μm on a cryostat (Leica CM1900). Brain sections were blocked with 10% NGS and 0.3% Triton-X in PBS for 1 h at room temperature, and then incubated with primary antibody (rabbit anti-Fos, SYSY system, 226 003, 1:10,000, RRID:AB_2231974; mouse anti-CamKII, Abcam, ab22609, 1:500, RRID:AB_447192; mouse anti-GAD67, Millipore, MAB5406, 1:1,000, RRID:AB_2278725) at 4 °C for 48 h. Sections were then washed with PBS for three times and incubated with secondary antibody (Alexa Fluor 488 goat anti-rabbit, Life Technologies, a11008, 1:2000, RRID:AB_143165; Alexa Fluor 555 donkey anti-rabbit, Life Technologies, a10040, 1:2000, RRID:AB_2534016; Alexa Fluor 488 goat anti-mouse, Life Technologies, a11029, 1:2000, RRID:AB_138404) for 2 h at room temperature. Sections were mounted and imaged with an Olympus VS120 microscope. For detection of Fos in ChR2 or GFP control mice, animals received 10-min of 20-Hz, ~5 mW photostimulation (488 nm) and were perfused 30 min later. For detection of Fos in mice with different aversive stimuli, mice were perfused 1.5 h after injection (i.p.) with different regents or electric foot shock unless otherwise mentioned.

Staining of embryos and larvae was identical. Antibodies were used at the following dilutions: rabbit anti-dsRed (1:400, Clontech 632496), rat anti-tropomyosin (1:200, Abcam ab50567), mouse anti-GFP (1:50, Developmental Studies Hybridoma Bank GFP-G1), mouse anti-integrin βPS (1:50, Developmental Studies Hybridoma Bank CF.6G11), mouse anti-Fascin (1:25, Developmental Studies Hybridoma Bank sn 7C, generous gift from Tina Tootle), and mouse anti-αTubulin (1:200, Sigma-Aldrich T6199). Conjugated fluorescent secondary antibodies used were Alexa Fluor 555 donkey-anti-rabbit (1:200), Alexa Fluor 488 donkey-anti-rat (1:200), and Alexa Fluor 647 donkey-anti-mouse (1:200) (all Life Technologies) and Alexa Fluor 488 donkey-anti-mouse (1:200, Life Technologies). Furthermore, Acti-stain 555 phalloidin (1:400, Cytoskeleton PHDH1-A) and Hoechst 33342 (1 μg/ml) were used on larvae. Embryos and larvae were mounted in ProLong Gold (Life Technologies, P36930).