Alexa fluor 488 conjugated goat anti rabbit igg h l antibody

Paraffin-embedded coronal brain sections (2 μm thick) were deparaffinized and rehydrated using standard protocols. For antigen retrieval, the sections were immersed in 10 m M sodium citrate buffer, pH 6.0 (Iatron) and boiled at 100 ° C for 10 min and cooled to RT. After blocking with 10% nonfat dry milk in 1× PBS, the sections were incubated overnight at 4 ° C with a mixture of rabbit polyclonal anti-CRISPLD2 antibody (1: 100 dilution; Abgent, San Diego, Calif., USA) and mouse monoclonal anti-GFAP (glial fibrillary acidic protein) antibody (1: 200 dilution; Sigma-Aldrich, St. Louis, Mo., USA). After washing, the sections were incubated with a mixture of Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) antibody (Life Technologies, Carlsbad, Calif., USA) and Alexa Fluor 568-conjugated goat anti-mouse IgG (H+L) antibody (Life Technologies) for 1 h at RT. After washing, nuclei were stained with 4′-6-diamidino-2-phenylindole (Life Technologies). The sections were then washed again and mounted. The mounted sections were observed under an LSM710 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
For competition experiments, prior to immunostaining, anti-CRISPLD2 antibody was preabsorbed with an excess amount of CRISPLD2 antibody blocking peptide (BP5570a; Abgent).