Alexa 555 conjugated donkey anti rabbit

C. elegans serotonin antibody staining was performed using the tube fixation protocol (McIntire et al., 1992 (link)). Briefly, synchronized young adult hermaphrodites were fixed in 4% paraformaldehyde (PFA) for 18 hr, with β-mercapto-ethanol for another 18 hr, with 1 mg/ml collagenase (Sigma Aldrich, Merk, Darmstadt, Germany) for 90 min and incubated for 24 hr with rabbit anti-5HT antibody (1:5000; Sigma Aldrich). Alexa 555-conjugated donkey anti-rabbit (1:500; Molecular probes) was used as secondary antibody.
For mouse immunohistochemistry, freshly isolated E11.5 embryos from C57Bl/6JRccHsd were fixed by immersion in 4% PFA. Rabbit anti-Sall2 (1:100; Sigma Aldrich), goat anti-Brn2 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-5HT (1:5000; Sigma Aldrich) and goat anti-5HT (1:200; Abcam, Cambridge, UK) antibodies were used. As secondary antibodies Alexa 555-conjugated donkey anti-rabbit and anti-goat, and Alexa 488-conjugated donkey anti-rabbit and anti-goat were used (1:600; Molecular probes, Invitrogen, Eugene, OR). Immunofluorescence samples were analyzed and photographed using a confocal TCS-SP8 Leica microscope.

The six cell lines previously mentioned were plated on 8-well chamber slides (Nalge Nunc) in RPMI medium containing 10% FBS. The cells were then rinsed and fixed in 4% paraformaldehyde for 30 minutes at room temperature. After permeabilization in 0.2% Triton X-100 for 10 minutes at 4°C and blocking with 5% bovine serum albumin and 3% donkey serum in PBS for 1 hour at room temperature, the cells were incubated with dysadherinmAb NCC-M53 antibodies, diluted 1:1000, overnight at 4°C. Antigens were visualized using Alexa-488-conjugated donkey anti-mouse and Alexa-555–conjugated donkey anti-rabbit (1:300, 30 minutes) secondary antibodies (Invitrogen). Nuclear staining was conducted with DAPI (Invitrogen). Images were acquired with a Nikon Eclipse E800 upright microscope and attached RetigaEXi CCD digital camera. Images were processed and analyzed using Adobe Photoshop 7.0.

Staining was performed on brain sections as described in our previous publication.5 In brief, sections were blocked by 10% normal donkey serum, and incubated in specific primary antibodies as follows: mouse anti‐ARRB2 (1:100 dilution), rabbit anti‐Iba1 (1:150 dilution) and rabbit anti‐TRAF6 (1:100 dilution). After being washed three times by PBS, the cells were incubated with corresponding Alexa 488‐conjugated donkey antimouse (1:200 dilution) and Alexa 555‐conjugated donkey anti‐rabbit (1:200 dilution) (Invitrogen, Gaithersburg, MD, USA) secondary antibodies. DAPI (Beyotime, Haimen, China) was used to stain nuclei. Images were obtained by confocal microscopy (Zeiss, Oberkochen, Germany). Quantification of the colocalization coefficient between ARRB 2 and TRAF6 displayed as Pearson coefficients in the colocalized volume (1, perfect correlation; 0, no correlation; −1, perfect inverse correlation).15