Supplementary file_Liver Gpr182 mCherry F4/80 co staining

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.

为分析mCherry报告基因荧光和F4/80染色，小鼠经二氧化碳麻醉后，通过左心室灌注20毫升磷酸盐缓冲盐溶液（PBS），随后以4%多聚甲醛（PFA）在PBS中进行灌注。灌注完成后，肝脏在4°C下用4% PFA固定16小时。固定后，肝脏至少用PBS清洗三次，并转移至含有30%（质量/体积比）蔗糖的PBS中，于4°C保存24小时。随后，取一段肝脏，将其嵌入最佳切割温度（OCT）复合物中，并在-80°C下保存，待进一步处理。使用低温切片机对OCT块进行切片，收集150微米厚的切片，放入含有PBS的2毫升试管中，并再次用PBS清洗三次。随后，切片用PBS三乙醇胺0.5%马血清5%进行渗透和封闭，以温和摇动的方式在室温下孵育1小时。之后，用含有0.3%三乙醇胺和5%马血清的抗体稀释液对切片进行三次清洗，每次5分钟。随后，在4°C下用抗体稀释液对切片进行过夜孵育，以抗F4/80鼠单克隆抗体（1:100；Biorad MCA497R，克隆Cl:A3-1）。孵育后，用抗体稀释液对切片进行五次30分钟的清洗。接着，在含有4′,6-二氨基-2-苯基吲哚（DAPI，1:1,000）的抗体稀释液中，与AlexaFluor-488偶联的二级抗体（1:500，Thermofisher，A21208）在4°C下过夜孵育。再次用抗体稀释液进行五次30分钟的清洗。通过在ScaleSQ2中孵育1小时（37°C，Hama等人）和随后在室温下用ScaleS4溶液孵育1小时，并温和摇动（Hama等人），对切片进行透明化处理。然后，将切片用ScaleS4溶液固定在显微镜载玻片上，并盖上盖玻片。使用配备LD LCI Plan-Apochromat 63x/1.2 Imm Korr DIC M27物镜的Zeiss激光扫描显微镜（LSM）880共聚焦显微镜对切片进行成像。为了重建3D体积图像，记录了多个z-stack（0.49微米体素深度），以覆盖总共30微米的深度。随后，使用FIJI分布的ImageJ（v1.53f51）对图像进行处理和3D渲染。参考文献：Hama H，Hioki H，Namiki K，Hoshida T，Kurokawa H，Ishidate F，Kaneko T，Akagi T，Saito T，Saido T，Miyawaki A. ScaleS：一种用于生物成像的光学透明化试剂盒。Nat Neurosci. 2015年10月；18(10)：1518-29. doi: 10.1038/nn.4107. Epub 2015年9月14日. PMID: 26368944。