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DAVID LEÓN NAVARRO Y COLS. AN. R. ACAD. NAC. FARM.

For the immunocytochemical detection of P2X7 and P2Y1,
cells were incubated with primary antibodies that recognized the
specified rat proteins: rabbit anti-P2X7 (1/100) and rabbit anti-P2Y1
(1/100) (Chemicon International, Temecula, CA, USA); mouse anti-
synaptophysin (1/500). As secondary antibodies were used: goat anti-
mouse IgG fluorescein conjugated (1/500), goat anti-rabbit IgG
rhodamine conjugated (1/500).

Immunocytochemical detection of phospho-CaMKII and total
CaMKII in granule neurons stimulated with L-Glu (100 µM, 5 min)
was carried out following procedures described previously (6).
Primary antibodies used in this work recognized the specified rat
proteins: rabbit anti-phospho-CaM-kinase II a/b (Thr286/287) (1/100)
(Upstate Charlottestville, VA, USA) and mouse anti-b-CaM-kinase II
(1/100) (Zymed Laboratories, San Francisco, CA, USA).

In all cases, incubations with primary and secondary antibodies
were done at 37 ºC for 1 h. In every experiment incubation with the
control antigen was made following the manufacturer´s instructions.
Controls were performed following the same immunocytochemical
procedure but replacing primary antibodies by the same volume of
PBS-BSA solution.

Immunofluorescence images were captured digitally using a
Kappa DX2 camera controlled by Kappa Image Base Control
software. For each culture, acquisition parameters were adjusted in
order to prevent brightest fluorescence from saturating the mean
intensity of granule neuron. These conditions were maintained with
the subsequent images. Three or four images under specific
conditions were captured in each one of the cultures. Fluorescence
quantification was performed as previously described (6).

2.4. Statistical analysis

Data are presented as mean ± SEM, and differences were tested
by one-way ANOVA with Bonferroni´s post test using GraphPad
Prism version 4.00 for Windows (GraphPad Software, San Diego,
CA, USA).

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