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Description
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For analysis of mCherry reporter fluorescence and F4/80 staining, mice were sacrificed by CO2 and perfused with 20 mL PBS through the left ventricle, followed by 4% PFA in PBS. After perfusion, the liver was postfixed in PFA 4% for 16 h at 4°C. After fixation, the liver was washed at least three times with PBS and transferred to PBS containing 30% (wt/vol) sucrose at 4 °C for 24 h. A piece of liver was then embedded in optimal cutting temperature (OCT) compound and stored at −80 °C until further processing. OCT blocks were sectioned using cryostat, and 150 µm sections were collected in 2 mL tubes containing PBS and further washed 3 times more with PBS. Sections were then permeabilized and blocked with PBS triton 0.5% Horse serum 5% for 1h with gentle rocking. After three washes with antibody diluent (PBS containing 0.3% Triton X-100 and 5% horse serum) for 5 min each, sections were incubated for 2 overnight at 4°C with anti F4/80 rat monoclonal antibody (1:100; Biorad MCA497R, clone Cl:A3-1) in antibody diluent. After five washes of 30 min each with antibody diluent, sections were incubated for 2 overnight at 4°C with AlexaFluor-488 conjugated secondary antibodies (1:500, Thermofisher, A21208) in antibody diluent containing 4′,6-diamidino-2-phenylindole (DAPI, 1:1,000). After five washes of 30 min each with antibody diluent, sections were cleared by incubation for 1 hour in ScaleSQ2 at 37°C (Hama et al.) followed by 1 hour in ScaleS4 solution at room temperature with gentle rocking (Hama et al.). The sections were then mounted to microscope slide with ScaleS4 solution and covered with a coverslip. The sections were imaged with a Zeiss laser scanning microscope (LSM) 880 confocal microscopes equipped with a LD LCI Plan-Apochromat 63x/1.2 Imm Korr DIC M27 objective. In order to reconstruct 3D volumetric images, several z-stack (0.49 µm voxel depth) were recorded to span a total of 30 µm deep. Images were then processed and 3D-rendered with FIJI distribution of ImageJ (v1.53f51). References Hama H, Hioki H, Namiki K, Hoshida T, Kurokawa H, Ishidate F, Kaneko T, Akagi T, Saito T, Saido T, Miyawaki A. ScaleS: an optical clearing palette for biological imaging. Nat Neurosci. 2015 Oct;18(10):1518-29. doi: 10.1038/nn.4107. Epub 2015 Sep 14. PMID: 26368944.
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