Dako high ph antigen retrieval system

Mouse eyes were fixed with 2% paraformaldehyde for 2 hours. After paraffin embedding, the eyeballs were cut into 5‐μm‐thick sections and mounted on charged glass slides. Slides were de‐paraffinized and subjected to citrate‐based antigen retrieval. Paraffin sections were re‐treated with the DAKO high pH antigen retrieval system (DAKO, Carpinteria, CA) using a domestic 600 kW microwave oven. Nonspecific antibody binding was blocked by incubating sections in 4% BSA followed by 10% non‐immune goat serum (Zymed Corp., San Francisco, CA). Primary antibody was applied at a 1:200 to 400 dilutions overnight at room temperature. Sections then were incubated with secondary antibody for 30 minutes. The localization of target proteins was demonstrated with pre‐diluted streptavidin‐horseradish peroxidase (Zymed) and 0.05% 3, 3‐diaminobenzidine in TBS, with H₂O₂ as the substrate. All sections were counterstained lightly with haematoxylin.

Mouse eyes were fixed with 2% paraformaldehyde (Agar Scientific Ltd. Cambridge, UK) for 2 hr. After paraffin embedding the eyeballs were cut into 5 μm thick sections and mounted on charged glass slides. Slides were de‐paraffinized and subjected to citrate‐based antigen retrieval. Paraffin sections were retreated with DAKO high pH antigen retrieval system (DAKO, Carpinteria, CA) using a domestic 600 kW microwave oven. Nonspecific antibody binding was blocked by incubating sections in 4% BSA, followed by 10% nonimmune goat serum (Zymed Corp., San Francisco, CA). Primary antibody was applied at a 1:200 to 400 dilutions overnight at room temperature. Sections then were incubated with secondary antibody for 30 min. The localization of target proteins was demonstrated with pre‐diluted streptavidin‐horseradish peroxidase (Zymed, UK) and 0.05% 3, 3‐diaminobenzidine in TBS, with H₂O₂ as the substrate. All sections were counterstained lightly with hematoxylin.