Figure 5
Immunofluorescence 3D imaging with sMAX media. (a) Quantification of fluorescence signal changes by the mix with MAX media. Fluorescence-labeled antibody was diluted 10 times with PBS, iMAX, and sMAX, and the fluorescence signal intensities were measured with fluorometer 18–24 h later. (b) Reconstituted 3D image of 2-mm brain slice. Samples is stained with tyrosine hydroxylase (TH), and then immersed in sMAX (b, left). Scale bars, 1 mm for whole image and 100 μm for ROIs. Visualization of blood vessels (stained with Laminin) and surrounding glial cells (stained with glial fibrillary acidic protein, GFAP) using immunostaining (b, right). Scale bar, 20 μm. (c) Visualization of collagen type I (green) in the breast cancer biopsy specimen. Nuclei are counter-stained with propidium iodide (red). Scale bar, 20 μm (d) Clearing of human cerebral organoids in sMAX media. Bright-field images of cerebral organoids exhibiting neuromelanins (d, left). Images were taken with an EVOS microscope (Thermo Fisher Scientific). Scale bar, 500 μm for whole cerebral organoid images and 50 μm for high-magnified images. 3D whole-mount immunostaining of organoids labeled for Vimentin (d, middle). Scale bar, 500 μm. 3D highly magnified image showing cellular arrangements stained with NESTIN (d, right). Scale bar, 20 μm. All fluorescent images were acquired using a TCS SP8 confocal laser-scanning microscope and processed with Las X software.
