Antigenfix

CLEM was performed using a workflow as described by Urwyler et al., (2015) (link). In brief, mice were intravenously injected with 20 μg of CD31-conjugated antibody to stain sinusoid blood vessels. After 5 min, mice were sacrificed and inferior vena cava were cannulated and livers were perfused (4 mL/min) with Antigenfix (Diapath) for 5 min at room temperature. After excision, 2-3 mm slices of livers were further fixed by immersion in Antigenfix for 1 h at 4°C, washed in PBS and sliced making use of Leica Vibratome VT1200S. Slices were first used for Near Infra-Red Branding to indicate a region of interest (ROI), making use of a Zeiss LSM780 with a MaiTai laser (Bishop et al., 2011 (link)). Next, high resolution confocal stacks were obtained with a Zeiss LSM880 FastAiry. The sample was then processed for volume EM by fixation with Karnovsky buffer and en bloc heavy metal staining as described by Steeland et al., (Steeland et al., 2018 (link)). The sample was mounted onto an aluminum pin and trimmed for imaging with a Zeiss Merlin with 3View2 (Gatan). Overlays of confocal and volume EM datasets was performed based on blood vessel staining, using the ec-CLEM plugin of ICY in order to identify Kupffer cells or monocytes (Paul-Gilloteaux et al., 2017 (link)). The EM datasets were segmented in MIB (Belevich et al., 2016 (link)) and 3D rendering was done in iMaris.

Animals were transcardially perfused with AntigenFix (Diapath) at P14–15. Their brains were then removed and postfixed in AntigenFix for 24 h before they were sectioned (100 μm) with a vibratome (Leica VT 1000 S) and processed for immunohistochemistry as free-floating sections. The mCherry signal was amplified using an anti-RFP rabbit antibody (1/1000, Rockland). Sections used for electrophysiology were fixed overnight in AntigenFix before biocytin was revealed with Alexa647-conjugated-streptavidin (1/200, Jackson ImmunoResearch). In all cases, sections were incubated with antibodies for 72 h at room temperature to increase antibody penetration into the tissue. Sections were counterstained with Hoechst (Thermo Fisher, 1/1000) and mounted in Fluoromount (Thermo Fisher).