Chicken anti egfp

We first treated brain sections with 1 mM cupric sulfate (CuSO₄) in 50 mM ammonium acetate buffer for removing autofluorescence. Then, sections were incubated for 1 h in 10% normal bovine serum (Jackson, West Grove, PA, USA) with 0.5% Triton X-100 for blocking unspecific staining. Next, the samples were incubated overnight at room temperature with chicken anti-EGFP (Invitrogen, 1:100, Carlsbad, CA, USA) and rabbit anti-NeuN (Millipore, 1:300, Darmstadt, Germany) antibodies diluted in PBS containing 0.5% Triton X-100. The sections were washed in PBS and incubated with secondary antibodies, donkey anti-rabbit IgG (Alexa Fluor 633-conjugated; 1:100; Invitrogen) and goat anti-chicken (IgG Alexa Fluor 555-conjugated; 1:100; Invitrogen, Carlsbad, CA, USA) for 1 h. This study employed Hoechst 33342 reagent for labeling cells nuclei. Finally, the brain sections were washed in PBS, mounted in fluoromount Vectashield (Vector Laboratories, Burlingame, CA, USA) and coverslipped for analysis by laser-confocal microscopy (Leica TCS SPE Microsystems GmbH, Wetzlar, Germany). Immunohistochemical controls were performed by omission of either the primary or secondary antibodies (data not shown).

EGFP
expression from primary neuronal and retinal transfected cells
was examined 72 h after their exposure to nanodiaplexes to qualitatively
assess the transfection efficiency by immunocytochemistry.^{36 (link)} Briefly, cover slips were incubated overnight
with chicken anti-EGFP (Invitrogen, 1:300). Cells were incubated for
1 h with secondary antibody Alexa Fluor555 goat anti-chicken IgG (Invitrogen,
1:100) which was pseudocolored in green to visualize EGFP expression.
Nuclei were stained with Hoechst 33342 (Sigma-Aldrich, Spain). Confocal
images were obtained using a laser-confocal microscope (Leica TCS
SPE Microsystems GmbH, Germany).