Tannic acid staining improves the visibility of tissues in the frozen blocks. (a–d) The eyeballs of adult ICR mouse were frozen and sliced for block-face observation. Without tannic acid staining, the retina (arrows) and lens (asterisk) are seen in white (a, b). By pre-staining with 1% tannic acid for 2 h, the retina is seen in brown with layered pattern, and the lens remains in white (c, d). The anterior and posterior spaces to the lens (aqueous and vitreous chambers) are seen in darker brown, which may reflect the remaining tannic acid solution or the shadow. (e, f) A section corresponding to the block face in d was stained with H&E, and used to identify the retinal layers; the retinal ganglion cell layer (RGCL), the inner plexiform layer (IPL), the inner nuclear layer (INL), the outer plexiform layer (OPL), the outer nuclear layer (ONL), inner and outer stripe of the photoreceptor layer (IS/OS), and retinal pigment epithelium (RPE). (g, h) The eyeball of adult C57BL5 mouse was imaged using CoMBI-S, and generated 919 block-face images with a voxel size of 2 × 2 × 4 µm. The reconstructed axial and equator planes (g) show retinal layers. The yellow and cyan lines indicate the positions of reconstructed axial and equator planes, respectively. 3D volume-rendered image (h) also shows the retina with some layers (arrows), and the lens (asterisk). (i–l) Mouse embryos on E10 were stained with tannic acid with indicated concentrations and staining times, then frozen. The block-face images shows that the visibility of tissues in the head region was improving, as increasing the concentration and staining time. The developing eyes in the upper panel (boxed areas) are enlarged in the lower panel. Bars in (a–h) 1 mm for whole eyeball, and 100 µm for magnified images, (i–l) 500 µm in the upper panel, 100 µm in the lower panel.
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