Dmi8 fluorescence microscope

For immunofluorescence experiments, cells were plated on coverslips, washed twice with PBS and fixed in 2% paraformaldehyde (PFA)/PBS for 20 min at room temperature. Cells were permeabilized in 0.5% Triton X-100/PBS for 10 min on ice and blocked with PBS 10% of goat serum for 1 h at room temperature. Primary antibodies were diluted in blocking solution overnight at 4 °C. Alexa Fluor 594- or 488-conjugated secondary antibodies were incubated for 1 h at room temperature. Nuclei were stained with DAPI (1:5000) for 5 min at room temperature and coverslips were mounted in Prolong Gold Antifade mountant (ThermoFisher, MA, USA). Conventional immunofluorescence was performed in a widefield DMi8 Leica fluorescence microscope and images analyzed in Las X software (Leica Microsystems, Germany). All image acquisitions were performed at 63×, 40× or 20× magnification and under the same exposure, gain and contrast conditions. Cells were separated into two groups: predominantly nuclear (N > C) or equal/ predominantly cytoplasmic (N ≤ C) and quantified according to [16 ].

The decreased mitochondrial membrane potential is a hallmark of early apoptosis. Following the manufacturer’s instructions (C2003S, Beyotime, China), IPEC-J2 cells were stained with JC-1 for 20 min at 37 ℃. A DMi8 Leica fluorescence microscope was used for visualization of JC-1 staining. Red fluorescence (high potential) indicated JC-1 aggregates in the matrix of mitochondria to form polymers (J-aggregates); green fluorescence represented that JC-1 was concentrated in the cytoplasm, and JC-1 was a monomer (low potential).

Mouse embryonic fibroblast NIH/3T3 cells (ATCC, CRL-1658) were seeded on 48-well plates, with a density of 20,000 cells per well, in Dulbecco’s Modified Eagle Medium (DMEM, Gibco 12,800–017), supplemented with 10% fetal bovine serum (SFB; Capricorn Scientific FBS-HI-11A) and 1% penicillin G-streptomycin (Gibco 15,140–122) at 37 °C under a humid atmosphere at 5% CO₂. Once 80% confluence was reached, cells were treated with 10, 100, and 500 μg/mL of each CD’s formulation for 24 h. After treatment, cells were incubated with 2 µM Calcein-AM (Invitrogen C3100MP) and 1.5 µM propidium iodide (PI, Sigma P4170) in DMEM without phenol red for 30 min at 37 °C. Calcein-positive cells, stained in green, correspond to live cells, while red nuclei are dead cells. Cells were immediately imaged using a 20 × objective with a Dmi8 Leica fluorescence microscope and analyzed by automatic counting with the Analyze Particles tool of the ImageJ software (NIH, USA). Viability was calculated as the percentage of live cells normalized against the total cell number (calcein + PI cells). Plots and statistical analysis were performed with the GraphPad Prism software v8.0, with results obtained from n = 4 independent experiments.

After the CHs were cultured and treated, they were washed with PBS three times. The mitochondrial membrane potential changes of CHs were measured using a JC-1 mitochondrial membrane potential detection kit (Solarbio, Beijing). The fluorescence changes and intensity of JC-1 monomers and multimers in CHs were detected using a DMi8 Leica fluorescence microscope (Leica, Germany).

In situ hybridization (ISH) was performed on paraffin-embedded zebrafish retina sections. Clrn1 transcripts were detected using an automated Leica Bond platform with heat-induced Leica ER2 antigen retrieval buffer solution and RNAscope 2.5 LS Protease III digestion (Advanced Cell Diagnostics, Hayward, CA, USA), as previously described (32 (link)). Briefly, after hybridization to 20ZZ probes targeting the region comprising nucleotides 2–911 of Dr-clrn1 NM_001002671.1, a six-step amplification process was performed, followed by chromogenic detection using Fast Red (Advanced Cell Diagnostics). Images were collected with a fully automated widefield DMi8 Leica fluorescence microscope.

All confocal images were acquired using a Leica SP8 or a Leica STELLARIS 5 confocal microscope equipped with a white light laser and 10×/0.45 C-Apochromat (HC PL APO CS2), 20×/0.75 (HC PL APO CS2), 25×/0.95 (HC FLUOTAR L VISIR), or 63×/1.20 (HC PL APO) objective. The images were obtained at room temperature using Leica LAS X software. All images were processed using Fiji ImageJ (National Institutes of Health) software. Each image represents maximum intensity projection of a Z-stack (capturing the entire lymphatic vascular layer, or the whole tissue Z-stack when imaging immune populations) of single tiles or multiple tile scan images. The ear tile scans in Fig. S1 were obtained using DMI8 Leica fluorescence microscope (Fig. S1 G) or Leica Thunder Imaging System (Fig. S1 H). The close-up images in Figs. 2, D and E; 3, A and C; 5 E; 6 H; 8 D; S2 F; and S4, E and F were additionally deconvolved using Huygens Essential (version 19.04; Scientific Volume Imaging) with theoretical point spread function and automatic background estimation. Details of image processing and quantification are provided in the supplemental material.