Zen 2.1 black software

RBCs were fixed and stained as described by Chakraborty et al. [16 (link)]. Briefly, cells were fixed in 4% (w/v) paraformaldehyde in 0.05 M phosphate buffer (PB, pH = 7.4) at 4°C for 60 min. After extensive washing with PB, they were permeabilized with 0.1% Triton X-100 for 20 min and incubated for 1 h in PB containing 1% bovine serum albumin and 10% normal goat serum to block nonspecific antibody binding. RBCs were immunolabelled with primary antibodies (single or double staining) at 4°C overnight. Incubations with the primary antibodies was followed by washing and incubation with goat anti-mouse Alexa®647- and/or goat anti-rabbit Alexa®488-conjugated secondary antibodies for 1 h at room temperature (for the antibody list, see supplementary material online, ).
After washing, RBCs were either processed for quantitative analysis (FACS, BD FACSCalibur™, BD Biosciences) [17 (link)] or were mounted in ImmunoHistoMount (Sigma-Aldrich, Saint Louis, Missouri, USA) and examined under a confocal laser-scanning microscope (Zeiss LSM 880, Axiocam 503 mono, 40x oil immersion objective, numeric aperture: 1.4; Carl Zeiss Microscopy GmbH, Germany). Eight-bit pictures were taken by using ZEN 2.1 (black) software (Carl Zeiss Microscopy GmbH 1997-2015) and analyzed by using ImageJ 1.51n and ZEN 2.1.

In an additional group of animals XCR1 in the spinal trigeminal subnucleus caudalis (Vc) was localized with the markers of glutamatergic synaptic terminals vesicular glutamate transporter 1 and 2 (VGlut1 and VGlut2, respectively). Naïve adult male Wistar rats (n = 3; 200–250 g) were deeply anesthetized and perfused transcardially with PFA. The brainstems were dissected out, post-fixed in the same PFA solution for 2 h at 4 °C and transferred into PBS containing 0.01% azide for a minimum of 24 h. Serial 50-μm transverse brainstem sections were incubated for 72 h at 4 °C in PBS containing rabbit anti-XCR1 (1:250; Abcam Cat# ab67342; RRID:AB_2217066; Abcam, Cambridge, UK) and guinea-pig anti-VGlut1 (1:2500; Millipore Cat# AB5905; RRID:AB_2301751; Merck Millipore, Germany) or guinea-pig anti-VGlut2 (1:5000; Millipore Cat# AB2251; RRID:AB_1587626; Merck Millipore, Germany) antibodies. Appropriate direct secondary antibodies were applied, sections were mounted with Gel Mount aqueous mounting medium (Sigma–Aldrich, UK) and visualized using a fluorescence microscope. Images were captured using an inverted confocal microscope (LSM 700; Carl Zeiss Microscopy, USA) in conjunction with Zen 2.1 (Black) software (Carl Zeiss Microscopy, USA).