Aec substrate kit

Human RPE cells on an eight-well Lab-TekTM chamber were fixed in 4% paraformaldehyde for 30 min, and then permeabilized using 0.2% Triton-X 100 at 37°C for 15 min. Blocking was achieved by addition of 1% goat serum for 20 min. The samples were incubated with primary anti-cleaved caspase-3 antibody (Cell Signaling; 1∶200) for 1 hr at room temperature. After washing with PBS, secondary biotinylated conjugated goat anti-rabbit antibody (1∶400; Vector, Burlingame, CA, USA) was applied to the slides for 30 min at room temperature. After washing with PBS, streptavidin peroxidase (Invitrogen, Camarillo, CA, USA) was applied to the slides for 30 min. 3-Amino-9-Ethylcarbazole (AEC) was added to the slide (AEC Substrate Kit, Invitrogen, Camarillo) which produced a red colored deposit. Sections were examined and photographed with microscope (Leica, Germany).

Immunoperoxidase staining was carried out following a previously described protocol [71 (link)] with some modifications. Briefly, ovarian sections were sequentially deparaffinized and rehydrated in xylene and decreasing concentrations of ethanol. Endogenous peroxidase was blocked by immersing sections in 3 % H₂O₂ in methanol for 30 min. Subsequently, the sections were immersed in 1 % glycine in 0.1 M PBS to block free aldehyde groups and then incubated in a blocking serum (10 % bovine serum albumin in 0.1 M PBS, pH 7.4) for 2 h at 4 °C to block non-specific bindings. Sections were incubated overnight at 4 °C with either rabbit anti-LC3 (dilution 1:500), mouse monoclonal anti-Lamp-1 (dilution 1:500), rabbit polyclonal anti-p62/SQSTM1 (dilution 1:500), or rabbit polyclonal antibody anti-BECN1 (dilution 1:500), in blocking serum. After washing with PBS, sections were incubated in secondary antibody, either goat anti-rabbit IgG-HRP (Abcam) or goat anti-mouse IgG-HRP (SouthenBiotech) at the dilution 1:500 in blocking serum, for 2 h at room temperature. Negative controls were performed by omitting the primary antibodies. The color was developed with an AEC substrate kit (Invitrogen). Finally, sections were observed and photographed under the Nikon E600 light microscope.