Eyeballs were fixed in dedicated eyeball fixed liquid (glacial acetic acid: formaldehyde: normal saline: ethyl alcohol=1:2:7:10) for at least 48 h. After gradient ethanol dehydration, paraffin embedding and other traditional sample disposal processes as previously described,33 (link),34 (link) 4 micron (μm) sections were cut through optic nerve in sagittal position. Then the sections were baked and dewaxed before hematoxylin and eosin (H&E) staining. After vitrification by dimethylbenzene and sealing by resinene, H&E staining sections were scanned with NanoZoomer Digital Pathology System (Hamamatsu Photonics Co., Ltd) and viewed with NDP view software. Then the thickness of cornea, retina and choroid were measured. The description of corneal thickness was from corneal epithelium layer to endothelial layer, and retinal thickness was from retinal internal limiting membranes to pigment epithelium layer. As for choroid thickness, it was from Bruch’s membrane (connected with retinal pigment epithelium layer) to sub-choroid space (attached to the sclera).
Protocol detail
Detailed Histological Evaluation of Ocular Tissues
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Bruch membrane
Choroid
Corneal
Corneal epithelium
Dehydration
Dimethylbenzene
Endothelial
Eosin
Epithelium
Ethanol
Eyeballs
Formaldehyde
Glacial acetic acid
Membranes
Normal saline
Optic nerve
Pigment
Retinal
Retinal pigment epithelium
Sclera
Vitrification
Corresponding Organization :
Other organizations : Southwest Jiaotong University, Chengdu Third People's Hospital, Air Force Medical University, Xijing Hospital
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Variable analysis
independent variables
- Eyeball fixation in a dedicated eyeball fixed liquid (glacial acetic acid: formaldehyde: normal saline: ethyl alcohol=1:2:7:10) for at least 48 h
- Thickness of cornea, retina and choroid
- Gradient ethanol dehydration
- Paraffin embedding
- Cutting 4 micron (μm) sections through optic nerve in sagittal position
- Baking and dewaxing of sections
- Hematoxylin and eosin (H&E) staining
- Vitrification by dimethylbenzene and sealing by resinene
- Scanning of H&E staining sections with NanoZoomer Digital Pathology System and viewing with NDP view software
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