Biology Reference
In-Depth Information
Table 2
Levels of EPO and S100E proteins in the serum and ACF of light-damaged rats
Serum levels (pg/mL)
ACF levels (pg/mL)
EPO
S100E
EPO
S100E
LD Ctrl
U (20)
U (10)
LD IM
90 ± 16 (10)
261 ± 24 (10)
12 ± 6 (10)
17 ± 2 (10)
LD SR
4 ± 2 (4/10)
6 ± 2 (3/10)
2400 ± 750 (9)
4550 ± 1370 (8)
ACF
anterior chamber fluid,
Ctrl
uninjected rats,
LD
light-damaged rats,
IM
intramuscular injection,
SR
sub-
retinal injection,
U
levels below the ELISA sensitivity which is 0.65 pg/mL for murine EPO and 0.33 pg/
mL for rat EPO. The measurements were performed at P65. The values are means ± SE, the number of eyes
analyzed is shown in brackects (
46
). Reproduced from Colella P. 2011 (
46
) by permission of Oxford
University Press
Move the cryostat arm backwards with the wheel so that the
following section results 10
m thick.
17. Continue sectioning and picking up each section in a serial way
(put the first section on slide 1, the second on slide 2, the 16th
section again on slide 1 and so on) (see Note 47).
18. When the first series of slides is completed, start filling in the
second series of slides (slides 15-30).
19. Check the quality of the sections under a dissecting micro-
scope every once in a while.
20. Air-dry the sections 1-3 h at room temperature.
21. Store the slides either at −20°C or at −80°C.
μ
1. Thaw two slides per eye and air dry them for 15 min at room
temperature.
2. Rehydrate the sections rinsing the slides twice in 1× PBS.
3. Put a few drops of 4¢,6-diamidino-2-phenylindole (DAPI) per
slide.
4. Cover the slides with cover-slips.
5. Fix the cover-slip passing a thin layer of transparent nail polish
on the coverslip borders.
6. Store the stained sections either at +4°C (short-term) or at
−20°C (long-term) in the dark.
3.8.2 DAPI Staining
of the Sections
1. Observe the eye sections stained with DAPI under a fluorescence
microscope.
2. Observe all the sections from each slide using a 5-10×
magnification.
3. Select three sections from the central part of the eye, which
have the largest globe diameter and contain the optic nerve
head (ONH).
3.8.3 Assessment
of Photoreceptor Nuclei
Rows
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