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VOL. 75 (4), 901-910, 2009 OVER-EXPRESSION OF P2Y2 RECEPTOR AFTER SILENCING...

and fixed with 4% paraformaldehyde in PBS 0.15M at 4 ºC for 6
hours. After fixation, corneas were embedded in Jung Tissue Freezing
Medium (Leica Microsystems, Barcelona, Spain) and 10 µm sections
were done. P2Y2 immunocytochemical assay was performed as
previously described for cells. Briefly, sections were permeabilized
with blocking solution (PBS 1X BSA 3% Triton X-100 FBS 5%) for
1 hour to block the non-specific binding, and after washing with
PBS 1X BSA 3%, sections are incubated with primary goat polyclonal
anti-P2Y2 (1:50) or PBS 1X BSA 3% for negative controls overnight
at 4 ºC. Sections were washed twice in PBS 1X BSA 3% and
incubated with the secondary antibody donkey anti-goat IgG-FITC
(1:200) for 1 hour at room temperature. Finally, after washing in
PBS 1X slices were mounting with Vectashield mounting medium
and observed under confocal microscope (Axiovert 200M; Carl Zeiss
Meditec GmbH, Jena, Germany), equipped with a Pascal confocal
module (LSM 5; Zeiss). All images were managed with the
accompanying Pascal software.

3. RESULTS

3.1. P2Y2 location in the cornea

Immunocytochemical analysis for P2Y2 in the cornea reveals that
this receptor is mainly localized in the outer layer of the epithelium
(Figure 1), while the inner layers of the epithelium are barely
marked. We have not found any P2Y2 signal in the other layers of the
cornea, neither in the stroma nor in the endothelium.

3.2. Inhibition of P2Y2 receptor expression by siRNA

After performing the treatments described in Methods, corneas
were wounded and the effect of the siRNA against the P2Y2 receptor
was tested by immunohystochemical analysis 12, 24 and 36 hours
after the healing (72, 84 and 96 hours after the first siRNA instilla-
tion). As we can observe, after the corneas were wounded, the P2Y2
receptor was still localized in the outer layer of the epithelium, being
this signal higher in control corneas than in Ap4A treated corneas,

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