All in one fluorescence microscope

Mouse eyeballs were enucleated and immediately fixed in 4% paraformaldehyde at 4°C overnight as previously described (Fu et al., 2018b (link)). After rinsed three times with PBS, the eyes were dehydrated and embedded in paraffin (Paraplast, Sigma-Aldrich, USA). Tissue slices (5 μm) were obtained using a rotation microtome (Thermo Fisher, USA), deparaffinized, and then rehydrated with graded ethanol for 5 min twice each. Antigen retrieval was conducted in citrate buffer. Once cooled, tissue sections were blocked with 10% goat serum and 0.2% Triton-X 100 in a dark and humid chamber for 2 h. After rinse briefly with PBS, the sections were immunolabeled with rabbit polyclonal antibody (α-smooth muscle actin, 1:100, Abcam) and incubated at 4°C overnight. After flushing, the samples were incubated with corresponding secondary antibodies (Alexa goat anti-rabbit 568, 1:500, Thermo Fisher) for 2 h. DAPI (Vector, CA) was used to visualize cellular nuclear. The slices were examined by the Keyence all-in-one fluorescence microscope (Itasca, USA) (Kasetti et al., 2016 (link)). For H&E staining, the paraffin section of mice TM tissues were sequentially deparaffinized, rehydrated, stained with hematoxylin and eosin (Sigma-Aldrich), dehydrated and sealed. The slices were visualized and photographed with phase contrast microscope (DMI 1, Leica).

Mice were anesthetized using 2% isoflurane on day zero, two, four, or seven after femoral bone injury. Femurs were removed, fixed in 4% paraformaldehyde, demineralized in 22.5% formic acid and 340 mM sodium citrate solution for 24 h, and embedded in paraffin. Immunostaining was performed as previously described [15 (link)]. To evaluate the number of osteoblastic cells at the bone surface, sections were incubated with an anti-F4/80 antibody (AbD Serotec, Raleigh, NC, USA) at a dilution of 1:20, followed by an incubation with an appropriate horseradish peroxidase-conjugated secondary antibody. Positive signals were visualized using the tyramide signal amplification system (PerkinElmer, Waltham, MS, USA), and sections were counterstained with 4′,6-diamidino-2-phenylindole and photographed using an All-in-One Fluorescence Microscope (KEYENCE, Osaka, Japan).

Whole-mount immunofluorescence staining of organoids was performed according to a previous protocol^{8 (link)}. Briefly, organoids were fixed and permeabilized using a Cytofix/Cytoperm Kit (BD Biosciences). The specimens were then incubated with primary antibodies [rabbit anti-lysozyme (1:10), mouse anti-mucin 2 (1:50), or mouse anti-Ki67 (1:50)] at 4 °C overnight. After washing three times with Perm/Wash Buffer, organoid specimens were further incubated with Alexa Fluor 568-conjugated anti-mouse IgG (1:800) or Alexa Fluor 647-conjugated anti-mouse IgG (1:100) for 3 h at 4 °C. After washing three times with Perm/Wash Buffer, the cells were incubated with DAPI (1:1000) for 10 min at RT. Fluorescence staining was visualized with fully focused z-stack images using an all-in-one fluorescence microscope (Keyence, Japan) equipped with structured illumination.