Model dmi8

At 3 hpf, zebrafish embryos were incubated with C6-PPNPs (50, 100, 200, or 400 ng/mL). After 5, 30, or 60 min, embryos were collected, rinsed with E3 medium, and assessed via fluorescence microscopy. Fluorescence images were obtained using a fluorescence microscope (Model DMi8, Leica, Germany). The microscope parameters were kept constant throughout the imaging process. Zebrafish C6-PPNP uptake at 7 days post-fertilization (dpf) was assessed via the same approach.

Mice were transcardially perfused with 0.9% saline. The mouse brains were fixed in 4% paraformaldehyde (PFA) for 72 h, followed by dehydration with different concentrations of ethanol and paraffin embedding. A paraffin microtome was used to cut 5-μm-thick coronal brain sections.
For immunofluorescence, sections were deparaffinized by ethanol and incubated with 3% H₂O₂. Antigen retrieval was performed by heating the sections in sodium citrate buffer (10 mM trisodium citrate, 0.5% Tween-20 in H₂O, pH 6.0) at 70°C for 30 min. Sections were then permeabilized and incubated with blocking solution containing the Iba-1 antibody (1:100, Abcam) overnight at 4°C. The sections were then stained with a fluorophore-conjugated secondary antibody (1:1,000, CST) for 1 h, followed by 4′6-diamino- 2-phenylindole (DAPI, Sigma) staining for another 1 h. Images were acquired by a fluorescence microscope Model DMi8 (Leica, Germany) and quantified using ImageJ software (National Institutes of Health) (Muhammad et al., 2019 (link)).

Frozen sections were prepared in the same way as for Nissl staining. Coronal sections 20 μm thick were stained according to the steps of the Iba-1 fluorescent staining instructions. The sections were fixed with 4% paraformaldehyde for 20 min, secondarily cleaned, and sealed with 10% goat serum for 30 min. Afterward, sections were permeabilized and blocked 48 h at 4 °C with Iba-1 antibody (1:100, Abcam, Cambridge, UK). A fluorophore-conjugated secondary antibody (1:200, ZSGB-BIO, Beijing, China) was applied to the sections for 2 h, followed by Dapi (Beyotime Biotechnology, Shanghai, China) staining for an additional 10 min. ImageJ v1.8.0 software (National Institutes of Health, Bethesda, MD, USA) was used to quantify images, acquired with a fluorescence microscope Model DMi8 (Leica, Wetzlar, Germany).

Fixed intestinal tissue was dehydrated and embedded in paraffin. Tissue sections (thickness of 4 μm) were stained with haematoxylin and eosin (HE, Olympus BX50; Tokyo, Japan). Observed by a Leica microscope (Wetzlar, Germany, ModelDMi8), 10 intact intestinal villi were randomly selected for each slice. Image-ProPlus (version 6.0) software was used to measure the height of each intestinal villus and its corresponding crypt depth and to calculate the ratio of the two. The height of the villi is defined as the vertical distance from the tip of the villi to the villi-crypt junction, and the crypt depth is the vertical distance from the villi-crypt junction to the base of the crypt.