Donkey anti mouse alexa 647

The relative localization of in the NI region of RLN3, serotonin and retrogradely-transported BDA in the NI region after MS injection was assessed by immunofluorescent detection of BDA – 5HT – RLN3. Briefly, free-floating brain sections were rinsed and incubated in blocking solution for 1 h and then transferred to primary antibody solution containing 1:2000 polyclonal rabbit anti-serotonin (S5545, Merck (Sigma-Aldrich); Madrid, Spain) and 1:5 mouse anti-RLN3 overnight at room temperature. For BDA – 5HT – RLN3 visualization, brain samples were incubated with 1:200 Alexa 488 donkey anti rabbit (#711–545-152, Jackson ImmunoResearch; Cambridge, United Kingdom), 1:200 Alexa 647 donkey anti-mouse (#715–605-150, Jackson ImmunoResearch; Cambridge, United Kingdom) and 1:200 Cy3 streptavidin (#016–160-084, Jackson ImmunoResearch; Cambridge, United Kingdom) for 2 h at room temperature. Finally, sections were mounted on clean slides and coverslipped using Fluorsave™.

Spleens, thymuses and Intestines were harvested fresh, directly embedded and frozen in OCT compound (Sakura, Torrance, CA), and 10 μm sections were cut at −20°C (Leica CM 3050 S). Frozen sections were left at RT to air dry for 5 min, fixed with cold acetone for 10 min (spleen) or incubated with 0.1 M PBS followed by 15 min postfixation in 4% paraformaldehyde in 0.1 M PBS (intestine), and subsequently washed 3 times with PBS for 10 min. Sections were incubated in blocking buffer (5% normal donkey serum and 0.8% Triton X-100) for 1 h at RT. Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C, washed 3 times with PBS for 10 min and then incubated with secondary antibodies diluted in blocking buffer as needed for 1 h at RT. Slides were washed 3 times with PBS 10 min each, air-dried and cover-slipped with ProLong Gold (Thermo Fisher Scientific, Carlsbad, CA), and imaged on a confocal microscope (Zeiss LSM-710). Anti-CD3 Alexa 488, Phalloidin Green 488, and anti-Podoplanin Alexa 488 were from Biolegend (San Diego, CA). Anti-B220 eFluor570 and donkey anti-goat Alexa 568 were from Thermo Fisher Scientific (Carlsbad, CA). Anti-pan neurofilament, anti-NF200, and anti-Contactin were from Neuromics (Edina, MN). Donkey anti-mouse Alexa 647 was from Jackson Immunoresearch (West Grove, PA).