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Exploration of the Visual System: Part 1: Dissection of the Mouse Eye for RNA, Protein, and Histological Analyses

互联网2013-12-31

2002

Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Due to the power of genetics, the mouse has become a widely used animal model in vision research. However, its eyeball has an axial length of only about 2 mm. The present protocol describes how to easily dissect the small rodent eye post mortem. This allows collecting different tissues of the eye, i.e., cornea, lens, iris, retina, optic nerve, retinal pigment epithelium (RPE), and sclera. We further describe in detail how to process these eye samples in order to obtain high?quality RNA for RNA expression profiling studies. Depending on the eye tissue to be analyzed, we present appropriate lysis buffers to prepare total protein lysates for immunoblot and immuno?precipitation analyses. Fixation, inclusion, embedding, and cryosectioning of the globe for routine histological analyses (HE staining, DAPI staining, immunohistochemistry, in situ hybridization) is further presented. These basic protocols should allow novice investigators to obtain eye tissue samples rapidly for their experiments. Curr. Protoc. Mouse Biol. 1:445?462 © 2011 by John Wiley & Sons, Inc.

Keywords: ophthalmology; visual sciences; retina; cornea; lens; iris; retinal pigment epithelium (RPE)

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Introduction
Basic Protocol 1: Enucleation of the Mouse Eye
Basic Protocol 2: Dissection of the Mouse Eye
Support Protocol 1: Preparation of Sylgard 184 Silicone Elastomer
Basic Protocol 3: RNA Preparation from Eye Tissues
Alternate Protocol 1: RNA Preparation from Pure RPE Cells
Basic Protocol 4: Protein Preparation from Eye Tissues
Basic Protocol 5: Fixation, Inclusion, and Cryosection of the Mouse Eye
Basic Protocol 6: Hematoxylin‐Eosin Staining
Basic Protocol 7: DAPI Staining
Basic Protocol 8: Immunohistochemistry
Support Protocol 2: Multiple Histochemical Stainings
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Enucleation of the Mouse Eye

Materials

Mice at the specific age needed for the study
70% (v/v) ethanol

IACUC‐approved animal housing facility
IACUC‐approved animal cages and bedding
IACUC‐approved food and water
Graefe forceps, 1‐mm tip, straight (Fine Science Tools)
Extra fine Bonn scissors, 8.5 cm, straight (Fine Science Tools)

Additional reagents and equipment for euthanizing the mice (Donovan and Brown, )

Basic Protocol 2: Dissection of the Mouse Eye

Materials

SYLGARD 184‐filled 60‐ or 100‐mm cell culture plates (see protocol 3 )
Eyeball (see protocol 1 )
Phosphate‐buffered saline (PBS; see recipe )
Cold‐light source, e.g., KL200 (Schott/Zeiss)

Stereomicroscope, e.g., Leica 16MZF, Leica M80, Zeiss Stemi 2000
Graefe forceps, 1‐mm tip, straight (Fine Science Tools)
0.2 mm Minutien pins, length 10 mm (Fine Science Tools)
Dumont #3c forceps (Fine Science Tools)
Vannas spring scissors 2‐ to 4‐mm blades (Fine Science Tools)
Extra fine Graefe forceps, 0.5‐mm tip, straight (Fine Science Tools)

Support Protocol 1: Preparation of Sylgard 184 Silicone Elastomer

Materials

SYLGARD 184 silicone elastomer kit (Dow Corning)
60‐mm and 100‐mm cell culture dishes
50‐ml conical polypropylene centrifuge tubes, with rim

Basic Protocol 3: RNA Preparation from Eye Tissues

Materials

Dissected eye ( protocol 2 )
RNA isolation solution, e.g., TRI Reagent (Molecular Research Center) or TRIzol (Invitrogen)
Chloroform
RNase‐free autoclaved water
RNase‐free 70% ethanol
cDNA synthesis kit, e.g., First‐strand cDNA synthesis kit for RT‐PCR (AMV; Roche)

RNase‐free 1.5‐ and 2‐ml microcentrifuge tubes
Ventilated chemical hood
18‐G Sterican needles (Braun)
1‐ml syringes (Braun)
FastPrep instrument (MP Biomedicals), optional
Lysing Matrix D tubes (MP Biomedicals), optional
Benchtop centrifuge, refrigerated

Alternate Protocol 1: RNA Preparation from Pure RPE Cells

Materials

Posterior eyecup ( protocol 2 , step 9)
SYLGARD 184‐filled 60‐mm or 100‐mm cell culture dish
Vannas spring scissors 4 mm blades (Fine Science Tools)
Trypsin
Phosphate‐buffered saline (PBS; see recipe )
Cell culture medium
RNA isolation solution, e.g., TRI Reagent (Molecular Research Center) or TRIzol (Invitrogen)
Chloroform
RNase‐free autoclaved water
RNase‐free 70% ethanol

37°C, 5% CO₂ incubator
Extra fine Graefe forceps, 0.5‐mm tip, straight (Fine Science Tools)
RNase‐free 2‐ml microcentrifuge tubes
RNase‐free 200‐µl pipet tips
Benchtop centrifuge, refrigerated

Basic Protocol 4: Protein Preparation from Eye Tissues

Materials

Ice
Tissue samples (see protocol 1 and 2 and the protocol 5 )
RIPA buffer (see recipe )
Tissue homogenization buffer (see recipe )

1.5‐ml microcentrifuge tubes
18‐G Sterican needles (Braun)
1‐ml syringes (Braun)
Plastic pestles adapted to 1.5‐ml microcentrifuge tubes
Microcentrifuge
Standard equipment for immunoblotting

Basic Protocol 5: Fixation, Inclusion, and Cryosection of the Mouse Eye

Materials

Eyeball (see protocol 1 )
Phosphate‐buffered saline (PBS; see recipe ), 1×
4% paraformaldehyde/1× PBS (see recipe )
30% sucrose/1× PBS (see recipe )
Embedding medium, e.g., according to Yazulla (see recipe )
Desiccating agent (e.g., drying pearls orange; Sigma)

1.5‐ml microcentrifuge tubes
Aluminum foil
Cryostat, e.g., Leica CM1900 (Leica)
SuperfrostPlus glass slides (Menzel)
Heating block, optional
Closed box for storing slides

Basic Protocol 6: Hematoxylin‐Eosin Staining

Materials

Slides with cryosections (see protocol 7 )
Phosphate‐buffered saline (1×, PBS; see recipe )
4% paraformaldehyde in 1× PBS (see recipe )
Hematoxylin solution, e.g., Accustain Hematoxylin solution, Gill No. 2 (Sigma)
70% ethanol/30% HCl 0.12 M solution (see recipe )
Eosin solution, e.g., Accustain Eosin Y solution, alcoholic (Sigma)
50% glycerol/50% 1× PBS solution (see recipe )
Nail polish

Staining station, optional
Coplin Jars or similar histology labware, optional
Coverslips (Menzel)
Light microscope

Basic Protocol 7: DAPI Staining

Materials

Slide with cryosections (see protocol 7 )
Phosphate‐buffered saline (PBS)
DAPI staining solution (see recipe )
Cityfluor AF3 mounting medium (Cityfluor)
Nail polish

Coverslips
Fluorescence microscope equipped with ultraviolet filter

Basic Protocol 8: Immunohistochemistry

Materials

Slides with cryosections (see protocol 7 )
Phosphate‐buffered saline (PBS)
Immunohistochemistry blocking solution (see recipe )
Primary antibodies (commercially available or cited in literature)
Secondary fluorescent antibodies (e.g., Molecular Probes, Invitrogen)
Cityfluor AF3 mounting medium (Cityfluor)
Nail polish

Immunohistochemistry tray chamber with lid (e.g., StainTray Slide Staining System, IHC world)
Coverslips
Fluorescence microscope equipped with appropriate filters

Support Protocol 2: Multiple Histochemical Stainings

Materials

Slides with cryosections ( protocol 7 )
Phosphate‐buffered saline (PBS), 1×
Immunohistochemistry blocking solution (see recipe )
Primary antibody
Secondary antibody coupled to Alexa Fluor 594 (Invitrogen)
DAPI staining solution (see recipe )
Fluorescein‐conjugated peanut agglutinin (FITC‐PNA; Sigma)
Cityfluor AF3 mounting medium (Cityfluor)
Nail polish

Immunohistochemistry tray chamber with lid (e.g., StainTray Slide Staining System, IHC world)
Coplin jars or similar histology labware, optional
21°C incubator
Coverslips
Fluorescence microscope equipped with appropriate filters

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Figures

Figure 1. Enucleation (, step 4). Pull the eyeball gently off the orbit, by pressing with the fingers around the orbit and pulling them apart.

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Figure 2. Enucleation (, step 5). Enucleate the eye by gently holding the eyeball with forceps and cutting the optic nerve with scissors at a distance of ∼2 mm from the eyeball.

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Figure 3. Dissection of the eye (, step 2). Hold the eyeball with forceps on the cell culture dish and immobilize with Minutien pins, pinning through the sclera and extraocular tissues close to the optic nerve (asterisk).

View Image

Figure 4. Schematic representation of the mouse eye. The size of the eyeball is ∼2 mm. The major components of the eye are indicated. Please note the reduced volume of the vitreous in the mouse eye. Abbreviations: RPE: retinal pigment epithelium.

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Figure 5. Dissection of the eye (, steps 5 to 7). After dissection of the mouse eye, several tissues can be isolated. The transparent lens (A ) often has iris tissue attached to the equator plane (asterisk). The yellowish retina (B ) too, may have attached iris tissue (asterisk). Dark pigmentation is further due to apical foldings of the RPE that are attached to the retina. The posterior optic cup comprises the melanized RPE and choriocapillaris, as well as the grayish sclera (C ). The white optic nerve extends form the eyeball (asterisk). With respect to the anterior segment, the melanized iris remains attached to the transparent cornea (D ).

View Image

Videos

Literature Cited

Literature Cited
	Braissant, O. and Wahli, W. 1998. A simplified in situ hybridization protocol using non‐radioactive labeled probes to detect abundant and rare mRNAs on tissue sections. Biochemica 1:10‐16.
	Chalupa, L.M. and Williams, R.W. 2008. Eye, Retina, and Visual System of the Mouse. The MIT Press, Cambridge, Mass.
	Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
	Eldred, W.D., Zucker, C., Karten, H.J., and Yazulla, S. 1983. Comparison of fixation and penetration enhancement techniques for use in ultrastructural immunocytochemistry. J. Histochem. Cytochem. 31:285‐292.
	Escher, P., Lacazette, E., Courtet, M., Blindenbacher, A., Landmann, L., Bezakova, G., Lloyd, K.C., Mueller, U., and Brenner, H.R. 2005. Synapses form in skeletal muscles lacking neuregulin receptors. Science 308:1920‐1923.
	Haverkamp, S. and Wässle, H. 2000. Immunocytochemical analysis of the mouse retina. J. Comp. Neurol. 424:1‐23.
	Smith, R.S., John, S.W.M., Nishina, P.M., and Sundberg, J.P. 2002. Systematic evaluation of the mouse eye: Anatomy, pathology, and biomethods. CRC Press, Boca Raton, Fla.
	Sugita, S. and Streilein, J.W. 2003. Iris pigment epithelium expressing CD86 (B7‐2) directly suppresses T cell activation in vitro via binding to cytotoxic T lymphocyte‐associated antigen 4. J. Exp. Med. 198:161‐171.
	Taft, R.A., Davisson, M., and Wiles, M.V. 2006. Know thy mouse. Trends Genet. 22:649‐653.