Biology Reference
In-Depth Information
3.7 Quanti fi cation of
EPO and S100E Protein
Levels in the Serum
and ACF of Rats
1. Thaw the serum and ACF samples (if stored at −80°C).
2. Use the Quantikine Mouse/Rat Erythropoietin ELISA.
3. Prepare all reagents, standard dilutions, and positive control as
described in the ELISA datasheet.
4. Measure two different dilutions of each sample.
5. Use 10-50
μ
L of serum and 0.1-10
μ
L of ACF (see Notes
41-44).
Table
2
shows the EPO and the S100E protein levels measured
in rats injected with AAV (
46
).
3.8 Evaluation of
Photoreceptor Survival
by Retinal Histology
1. Set the temperature of the main cryostat chamber at −26°C
and that of the specimen at −23°C.
2. Keep the mold containing the O.C.T.-embedded eye in the
cryostat chamber for 15 min to let the mold reach −26°C.
3. Remove the O.C.T. block from the mold, put a thin layer of
fresh O.C.T. on the specimen chuck, put the block on the chuck
so that the bottom of the block (where the eye is located) is in
front of you and is parallel to the chuck base (see Note 45).
4. Let the thin layer of O.C.T. solidify so that the block is firmly
fixed on the chuck.
5. Insert the chuck in the clamp and tighten the wing nuts to
hold it firmly.
6. Insert the blade into the blade holder.
7. Move the cryostat arm backwards with the wheel so that the
block is located behind the blade.
8. Try to align the flat bottom side of the block and the blade
edge so that they are parallel.
9. Remove the excess of O.C.T. surrounding the embedded eye
by hand using a trimming blade.
10. Set the section thickness at 20-30
3.8.1
Eye Cryosectioning
m and start trimming.
11. Pick up each section by leaning the slide towards the polarized
side of the section. The section will immediately stick to the
slide.
12. Observe the section under a dissecting microscope (see Note 46).
13. Once you achieve an eye section in which it is possible to dis-
tinguish the anterior (e. g. cornea and ciliary epithelium) from
the posterior (retina and RPE) part of the eye, you can stop
trimming and set the section thickness to 10
μ
m.
14. Number 30 slides (from 1 to 30) and mark the side of the slide
corresponding to the temporal side of the section with a
pencil.
15. Place the first series (1-15) on the cryostat in front of you.
μ
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