眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

The human eye is filled with a liquid extracellular matrix called vitreous in the mouse eye. This space is mostly filled by the lens. To isolate the vitreous and retina, we first have to dissect the anterior segment, stabilize the eye with forceps, and use a super sharp blade to make a single incision across the cornea.

A dull blade can tear the cornea and require a few strokes. A WEX cell sponge is used to absorb fluid in the anterior chamber. The arrows show where we'll squeeze the lens and vitreous out with forceps, partially close the forceps and pull forward.

The lens looks like a clear sphere and trailing behind it will be the vitreous gel. In this example, the vitreous remains adherent to the back of the lens to isolate the retina forceps are placed further back near the optic nerve, squeeze gently and pull forward. The retina appears like a yellow gelatinous material.

In some cases, the retina vitreous and lens will come out as a single tissue. The next step is to isolate and separate these three tissues using a filter centrifugation device. The filter piece goes in the top of an einor tube and we add some phosphate buffered saline.

As each of the tissues are isolated, they're placed in the top upper well containing the filter. The lenses in the upper left corner and the retina vitreous is in the lower right place. The tubes in a micro centrifuge and spin at 14, 000 Gs for 12 minutes.

Vitreous will spin to the bottom of the einor tube lens, and retina will stay in the upper. Well use forceps to separate the lens from the retina, then isolate the retina from the upper well. All these tissues can be placed in einor tubes and then processed for proteomics analyses.