Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

Our lab focuses on ocular development and the diseases.This protocol describes a method to remove the skull and the orbital bones sequentially from dissected mouse head.Leaving eyelids, ocular surface, lens, and the retina all together in one piece.The protocol will benefit ophthalmologists who are interested in ocular surface didactics.I am Dianlel Guo, to tell the technique with a likely struggle when trying to avoid the damaging of the eyeball, while dissecting the facial bones.The main advantage of this dissection matter is that the integrity of the ocular surface epithelial sheet is well preserved and the different parts of the ocular surface can be examined by histology or immunostaining in one single section.Be sure to cut off all small bones and debris off of the soft tissues.Reason, it will cause scar marks on tissue section otherwise.After euthanizing the mouse, use a pair of straight scissors to cut off the head and bisect it along the sagittal midline beginning at the intraparietal bone rostrally to the nasal.Place each half of the bisected head into a clean petri dish and use sharp scissors to cut off the jaw and tongue.Free the optic nerve by cutting it off the brain prior to the optic chiasm location.And remove all brain tissues including the olfactory bulb, which is attached to the skull wall.Now all of the remaining tissues shall have all of the skull and orbital bones associated with the eyeball.Wash the remaining parts with PBS to clean off any blood and hair debris.Pre-fix the tissues by placing them in a 50 milliliter tube containing 4%paraformaldehyde and rotating them at room temperature.After prefixation is complete, quickly wash the dissected head in PBS three times to further eliminate the broken hairs and fixatives.Place the tissue into a 10 centimeter petri dish and cut the skull into three parts along the planes indicated in figure one of the text protocol.The eyeball and the socket is hidden in the middle part of the skull under ethmoid, frontal, and maxillary bones.Use two pairs of number four straight forceps to peel off the ethmoid bone and fully expose the frontal and maxillary bones.Then, geographically divide the skull surface into four areas.Insert the tip of curved forceps horizontally into each area to remove the maxillary and frontal bones sequentially.After this, the underlying lacrimal and jugal bones will be exposed.Remove the two bones and associated subcutaneous muscles and fats surrounding the eyeball.Trim the eyeball with attached eyelids and skin into a small, square shaped block to reduce tissue volume and facilitate orienting the tissue when embedding.Place the eyeball and associated tissues back into 4%paraformaldehyde and continue to fix overnight at 4