Materials and Reagents

Eppendorf Tube (Eppendorf, catalog number: 0030 102.002)
Glass Pasteur Pipettes (Teklab Limited, UK catalog number: GP225)
Centrifuge tube, conical bottom, 13 mL volume (VWR, catalog number: VWRI525-0179)
Milli-Q^® Biocel System water (Millipore, http://www.millipore.com/)
Ampicillin (Millipore, catalog number: 171257), storage at -20°C
Protease inhibitor cocktail (Millipore, catalog number: 539133), storage at -20°C
NaCl (VWR, catalog number: 7647-14-5), room temperature storage
KCl (VWR, catalog number: 7447-40-7), room temperature storage
Na₂HPO₄ (Millipore, catalog number: 567547), room temperature storage
NaH₂PO₄ (Millipore, catalog number: 567545), room temperature storage
MgCl₂·6H₂O (Millipore, catalog number: 442615), room temperature storage
CaCl₂·2H₂O (Millipore, catalog number: 137101), room temperature storage
Steritop^® Bottle-top Filtration Units 0.22 μm (Millipore Express^® PLUS)
MitoTracker^TM Deep Red 633 FM Dye (ThermoFischer Scientific, catalog number: M46753)
Mammalian Ringer (MR) (see Recipes)
MitoTracker^TM Deep Red 633 FM Dye stock solution (see Recipes)

Equipment

Pipetting Standard (Gilson, Pipetman^®, models: P20, P200, and P1000) (http://www.gilson.com/Products/)
MicroCentrifuge (Eppendorf, model: 5417R)
Dissecting Scalpels and Blades, dissecting Scissors, dissecting dressing Forceps, as in United (Scientific^TM Dissecting Set, catalog number: S111010)
Dewar flask
Red light/dark room
Vacuum filtration device

Procedure

A flowchart depicting the whole experimental procedure is shown in Figure 2.

Figure 2. Flowchart of the procedure to detach the retina from the eye semi-cup with the MR incubation.

Prepare the eye semi-cup for incubation
Operations from step A2 on must be carried out at room temperature, in a dark room in dim red light (lower than 10 lux, 620 nm red lighting), to avoid rhodopsin bleaching. This facilitates the detachment of the photoreceptor OS from the RPE.
1. Obtain freshly enucleated bovine eyes from a slaughterhouse and use them within 2 h of animal death. The age of the cattle from which the eyes can be obtained for scientific purposes may be limited (as is the case in Italy) to 1.5 years, as a cautionary measure for the risk of possible transmission of Bovine Spongiform Encephalopathy (BSE). Eyes must be kept warm, in a light-tight container, possibly a Dewar flask, and handled to minimize the temperature decline.
2. Remove extraocular tissues with scissors.
3. Gently separate the anterior segment and vitreous from the eye semi-cup containing the neural retina. Make an incision in the sclera with a scalpel on one side of the eyeball, then hemi-sect the eye using scissors, approximately at the level of the ora serrata. Remove the anterior chamber, the lens, and vitreous carefully and discard them.
Detach the retina with MR incubation (Figure 3)
1. Fill the eye semi-cup containing the retina still attached to the RPE with 5–7 mL (depending on the size of the eye) of sterile filtered MR containing 2 mM glucose, protease inhibitor cocktail, and Ampicillin (100 μg/mL). All the operations, including incubations, are conducted at room temperature. The surface for dissection is routinely an aluminum foil.
2. Incubate each eye semi-cup for 10–15 min to allow the retina to detach completely. Eyes should be dissected and incubated with MR in rapid sequence.
Figure 3. CLSM evaluation of OS distribution in the retina and the eye semi-cup after asportation with MR. A) Mitotracker signal in a whole retina spontaneously detached from the eye semi-cup. In this case, a homogeneous layer of photoreceptors is recognizable. B) Mitotracker signal in an eye semi-cup from which the retina was let float to detach spontaneously from RPE. In this case, only mitochondria are visible (white head arrows). MitoTracker Deep Red 633 was used, dissolved in DMSO, and kept at -20°C in dark vials.
Gathering of Retinas (Figure 4)
1. After 10 min of incubation, rods spontaneously detach from the RPE, and the retina freely floats inside the eye semi-cup, attached to it through the optic nerve fibers; upon shorter incubation time, if it is necessary to remove the retina, gently shake the eye semi-cup by hand for 10 s, avoiding spilling the liquid contained inside, until the retina completely detaches from the RPE.
2. Rapidly turn the eye semi-cup inside out and discard the MR previously filling the semicup and now dripping out.
3. Cut each retina free from the optic nerve with scissors, letting the retina drop into a centrifuge 13 mL tube.
Figure 4. CLSM evaluation of OS distribution in the retina and the eye semi-cup after retina asportation with a soft brush. A) Retina detached from the eye semi-cup by a soft brush. In this case, the sample is characterized by a poor OS layer, allowing the visualization of the underlying mitochondria (white head arrow) belonging to inner segments of photoreceptors and the other retinas layers. B) Mitotracker signal in an eye semi-cup from which the retina had been detached with a soft brush. Specifically, it is possible to observe mitochondria (white head arrows) and OS immersed into the RPE (white arrows) since they have been torn from their ellipsoid.

Data analysis

The present method allows an increase of approximately 300% in the yield of total OS disk protein compared with the mechanical removal of the retinas from the eye semi-cup with a tweezer. When OS disks are purified by the method in Smith et al. (1975), the yield is 0.5 mg of rhodopsin/retina, corresponding to 0.58 mg protein/retina and approximately 11 mg total protein from 20 retinas (Smith et al., 1975). We have purified disks from 20 retinas utilizing the method described by Smith et al. (1975), extracting the retinas from the eye semi-cups, both following the present protocol and the mechanical method utilizing tweezers. The total disk protein yield, as assayed according to Bradford (Bradford, 1976), was 30 ± 2 mg of total disk protein in the first case and 10 ± 0.8 mg of total protein when retinas were extracted using tweezers.

Notes

In our experience of more than 10 years using this method, the reproducibility is excellent. Any eye of cattle up to 1.5 years of age (limitation due to BSE precaution rules) can be treated as described with predictable outcomes. On the other hand, if retinas are mechanically detached from the eye semi-cup, either with scissors or a brush, the yield in rod outer segment protein is extremely variable and can be approximately 300% lower. Additionally, our procedure maintains the physiological features of the retinal tissue.
Cautionary points:
(i) To prevent rhodopsin activation, avoid any white light source (McDowell and Kühn, 1977) by performing experiments in a dark/red light room;
(ii) Remove the vitreous humor from the eye semi-cup with special care, i.e., by gentle and rapid manual squeezing to avoid premature detachment of the retina, which would invalidate the present procedure;
(iii) Keep the eyes warm, minimizing the temperature drop, as eye cooling causes disaggregation of the RPE, which can contaminate the retinas, even when extracted with the present procedure;
(iv) As the bacterial dimension is similar to the rod disks (Smith and Litman, 1982), work in sterile conditions, filter sterilize the medium, use an antibiotic, and thoroughly clean the dissection tools with disinfectant soap and warm water.

Recipes

Mammalian Ringer (MR)
7.850 mL of 1 M NaCl
0.250 mL of 1 M KCl
1.750 mL of 200 mM Na₂HPO₄
2.000 mL of 200 mM NaH₂PO₄
0.025 mL of 1M MgCl₂
Mix 0.025 of 1 M CaCl₂ pH 6.9 in Milli-Q^® water; add the protease inhibitor cocktail, according to the manufacturer instructions), and 100 μg/mL of Ampicillin.
When MT Deep Red 633 FM Dye is necessary, add it form the stock solution at 500 nM final concentration.
MitoTracker^TM Deep Red 633 FM Dye stock solution
Dissolve the mitochondrial dyes MT Deep Red in dimethylsulfoxide (DMSO) to make a 200 µM stock solutions and keep the solution at −20°C in dark vials.

Acknowledgments

This work was supported by FRA (Fondi per la Ricerca di Ateneo) 2019 from University of Genoa. This protocol was derived from previous original research articles, Calzia et al. (2018) and Ravera et al. (2020).

Competing interests

Authors declare absence of financial and non-financial competing interests.

Ethics

The present protocol utilizes bovine eyes from cattle intended for human consumption, which are killed at local slaughterhouses; therefore, ethic issues can be considered ruled out.

References

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
Bruschi, M., Bartolucci, M., Petretto, A., Calzia, D., Caicci, F., Manni, L., Traverso, C. E., Candiano, G. and Panfoli, I. (2020). Differential expression of the five redox complexes in the retinal mitochondria or rod outer segment disks is consistent with their different functionality. FASEB Bioadv 2(5): 315-324.
Bubis, J. (1998). Effect of detergents and lipids on transducin photoactivation by rhodopsin. Biol Res 31(1): 59-71.
Calzia, D., Bianchini, P., Ravera, S., Bachi, A., Candiano, G., Diaspro, A. and Panfoli, I. (2010). Imaging of living mammalian retina ex vivo by confocal laser scanning microscopy. Anal Methods 2(11).
Campbell, L. J., West, M. C. and Jensen, A. M. (2018). A high content, small molecule screen identifies candidate molecular pathways that regulate rod photoreceptor outer segment renewal. Sci Rep 8(1): 14017.
de Grip, W. J., Daemen, F. J. and Bonting, S. L. (1972). Enrichment of rhodopsin in rod outer segment membrane preparations. Biochemical aspects of the visual process—XVIII. Vision Res 12(10): 1697-1707.
Gilliam, J. C., Chang, J. T., Sandoval, I. M., Zhang, Y., Li, T., Pittler, S. J., Chiu, W. and Wensel, T. G. (2012). Three-dimensional architecture of the rod sensory cilium and its disruption in retinal neurodegeneration. Cell 151(5): 1029-1041.
Masland, R. H. (2012). The neuronal organization of the retina. Neuron 76(2): 266-280.
McDowell, J. H. and Kuhn, H. (1977). Light-induced phosphorylation of rhodopsin in cattle photoreceptor membranes: substrate activation and inactivation. Biochemistry 16(18): 4054-4060.
Molday, R. S., Hicks, D. and Molday, L. (1987). Peripherin. A rim-specific membrane protein of rod outer segment discs. Invest Ophthalmol Vis Sci 28(1): 50-61.
Molday, R. S. and Moritz, O. L. (2015). Photoreceptors at a glance. J Cell Sci 128(22): 4039-4045.
Netter. (2018). Atlas of Human Anatomy. (Professional Edition). Including Student Consult Interactive Ancillaries and Guides. Elsevier.
Palczewski, K. (2014). Chemistry and biology of the initial steps in vision: the Friedenwald lecture. Invest Ophthalmol Vis Sci 55(10): 6651-6672.
Panfoli, I., Calzia, D., Ravera, S., Bianchini, P. and Diaspro, A. (2010). Immunochemical or fluorescent labeling of vesicular subcellular fractions for microscopy imaging. Microsc Res Tech 73(12): 1086-1090.
Papermaster, D. S. (1982). Preparation of retinal rod outer segments. Methods Enzymol 81: 48-52.
Raubach, R. A., Franklin, L. K. and Dratz, E. A. (1974). A rapid method for the purification of rod outer segment disk membranes. Vision Res 14(5): 335-337.
Ravera, S., Calzia, D., Bianchini, P., Diaspro, A. and Panfoli, I. (2007). Confocal laser scanning microscopy of retinal rod outer segment intact disks: new labeling technique. J Biomed Opt 12(5): 050501.
Smith, H. G., Jr. and Litman, B. J. (1982). Preparation of osmotically intact rod outer segment disks by Ficoll flotation. Methods Enzymol 81: 57-61.
Smith, H. G., Jr., Stubbs, G. W. and Litman, B. J. (1975). The isolation and purification of osmotically intact discs from retinal rod outer segments. Exp Eye Res 20(3): 211-217.
Stenkamp, D. L. (2015). Development of the Vertebrate Eye and Retina. Prog Mol Biol Transl Sci 134: 397-414.
Uhl, R., Desel, H., Ryba, N. and Wagner, R. (1987). A simple and rapid procedure for the isolation of intact bovine rod outer segments (ROS). J Biochem Biophys Methods 14(3): 127-138.
Young, R. W. (1967). The renewal of photoreceptor cell outer segments. J Cell Biol 33(1): 61-72.
Zimmerman, W. F. and Godchaux, W., 3rd (1982). Preparation and characterization of sealed bovine rod cell outer segments. Methods Enzymol 81: 52-57.