Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

Jessica M. Skeie; Stephen H. Tsang; Vinit B. Mahajan

doi:10.3791/2795

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

JoVE Journal
Biology
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JoVE Journal Biology

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

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02:39 min

April 3, 2011

DOI: 10.3791/2795-v

Jessica M. Skeie^1,2, Stephen H. Tsang³, Vinit B. Mahajan^1,2

¹Omics Laboratory,University of Iowa, ²Ophthalmology and Visual Sciences,University of Iowa, ³Harkness Eye Institute,Columbia University College of Physicians and Surgeons

Overview

This article details a dissection technique for isolating the vitreous, retina, and lens from the mouse eye. The process includes evisceration, centrifugation, and characterization using protein assays.

Key Study Components

Area of Science

Neuroscience
Biology
Ophthalmology

Background

The mouse eye contains a liquid extracellular matrix known as vitreous.
The anterior segment must be dissected to access the vitreous and retina.
Proper dissection techniques are crucial to avoid tissue damage.
Isolation of these tissues is important for further analysis.

Purpose of Study

To demonstrate a method for isolating ocular tissues from the mouse eye.
To facilitate subsequent proteomic analyses of the isolated tissues.
To provide a clear protocol for researchers in the field.

Methods Used

Dissection of the anterior segment of the mouse eye.
Use of forceps and a sharp blade for tissue extraction.
Centrifugation for tissue separation.
Characterization of isolated tissues with protein assays.

Main Results

The vitreous, retina, and lens can be successfully isolated.
Centrifugation effectively separates the tissues based on density.
The protocol allows for the collection of pure samples for analysis.
Visual aids enhance understanding of the dissection process.

Conclusions

This technique provides a reliable method for isolating ocular tissues.
It can be applied in various research contexts, including proteomics.
Further studies can build on this methodology for advanced analyses.

Frequently Asked Questions

What is the purpose of isolating the vitreous and retina?

Isolating these tissues allows for detailed analysis of their biochemical properties.

What tools are necessary for the dissection?

A sharp blade, forceps, and a WEX cell sponge are essential for the procedure.

How does centrifugation aid in tissue separation?

Centrifugation separates tissues based on their density, allowing for isolation.

Can this method be used for other species?

While this method is designed for mice, similar techniques may be adapted for other species.

What are the potential applications of the isolated tissues?

Isolated tissues can be used for proteomics and other biochemical analyses.

Is there a risk of damaging the tissues during dissection?

Yes, using a dull blade can cause tearing, so precision is important.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

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.