Obtain freshly enucleated bovine eyes from a local slaughterhouse within 1.5 hr of animal death. Carry out all operations at room temperature.
1 Cut eyes in half and divide the eyeballs into two eyecups. Carefully remove the cornea, lens and vitreous and discard. Perform these steps in dim red light.
CRITICAL STEP: The vitreous must be removed with great care, by gently squeezing the eyesemicup, in order not to detach retina while pulling it away.
2 Fill the semi-cup containing choroid and retina still attached to the RPE with 6-9 ml of MR (depending on the dimensions of the eye) containing 2 mM glucose, protease inhibitor cocktail and Ampicillin (100 μg/ml) and incubate for 5-10 min. Glucose is freely taken up by neurons of the retina thank to the presence of specific transportrs, such as GLUT -1 in rods6. If necessary to test the specificity of the chosen dye, add to MR aliquots of the appropriate inhibitor (for example, to test the specificity of Mitotracker staining, classical respiratory chain inhibitor such as: 10 μM Rotenone and 10 μM Antymicin A can be added to the solution inside the eyecup. CAUTION:These are harmful chemicals, carry out this step taking the necessary precautions.) Conduct this step in dim light.
3 Add mitochondrial vital dyes (for example: MitoTracker Deep Red 633(MT) and JC-1) from a stock solution, to the solution already present in the eye semicup in order to reach the desired final concentration (from 50 to 500 nM for MT). The staining solutions must be prepared upon use. Eye-semicups must not be washed. Operate and incubate in the dark for more 10-15 min.
4 Mount the sample for imaging: this step can be performed using option A or option B depending on whether it is preferred that retinas are detached from the RPE or left inside it.
a. After 20 min or more of incubation, rods spontaneously detach from pigmented epithelium so that the rim of the retina floats. Gently shake the whole eye semi-cup for 5 times until the retina is completely detached. Remove retinas from the eye semi-cup by rapidly turning it inside out and cut the retina free from the optic nerve.
b. Mount retinas onto the coverslip for CLSM. Do not wash nor fix the retinas. If necessary, add inhibitors (for example to test the specificity of JC-14 μM Nigericin, plus 5 μM Valinomycin can be added on the living retinas, on the coverslip during the during CLSM measurement. CAUTION: Nigericin and Valinomycin are toxic chemicals, take the necessary precautions)
a. If rods must be left embedded into the RPE, set the overall incubation time to no more than 10 min, then chose one of the two following options:
i. Cut at the rim all around the posterior eye semi-cup and mount it on the coverslip chamber with the face containing the retina downwards.
ii. Alternatively, immerse the whole semi-cup in a small pool arranged all over the immersion objective of the microscope. The objective must be carefully wrapped in a plastic glove and allow its tip to emerge through a small hole. By this set-up it is possible to hold the eye-semicup meanwhile the objective is free to move up and down in order to reach the proper focal planes. You need not to fix, nor wash samples.
5 To visualize results, image the labelled retinas. Perform CLSM imaging on the above samples, at 23°C. Acquire the measurements by means of a Leica TCS SP5-AOBS (Leica Microsystems, Mannheim, Germany) inverted confocal laser scanning microscope equipped with 457-476-488-514-543-633 nm laser lines.
Specimens are normally examined with an HCX APO L U-V-I 63x/0.9NA (Leica Microsystems,Mannheim, Germany) water immersion objective with a working distance of 2.2mm. This objective guarantees a good balance between confocal sectioning and penetration depth7. Either leave retinas in the eye-semicup or detach from the choroid. In the former case, cut the whole semicup at the rim and mount onto the coverslip chamber. In the latter case immerse the whole semicup in a small pool arranged around the objective. By this set-up it is possible to hold the eye-semicup while the objective is free to move up and down in order to reach the proper focal planes. This procedure is adopted when incubation is longer than 10 min, as in this case retina detaches spontaneously from the pigmented epithelium (RPE). No differences were noticed in either procedure.
Excite retinas stained with MitoTracker Deep Red 633 at 633 nm, and collect the emission in the spectral range from 650 to 700nm. Keep the laser power and the detection settings equal in all the experiments in order to avoid artefacts comparing retinas treated or not with inhibitors. Acquire, store and visulaize the resulting images with the Leica Confocal Software (LCS,Leica Microsystems, Mannheim, Germany). Carry out image elaboration, analysis and tridimensional rendering by ImageJ software (U. S. National Institutes of Health, Bethesda, Maryland,USA).