Biotin conjugated goat anti rabbit ig

Tissue sections were de-paraffined with xylene, and rehydrated through an ethanol series and TBS. Antigen retrieval was performed by microwave treatment, with Citrate buffer, pH6. Endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol for 30 min, followed by incubation with Protein Block (Genostaff) and avidin/biotin blocking kit (Vector). The sections were incubated with anti-LIGHT(TNFSF14) rabbit polyclonal antibody (SIGMA) at 4 °C overnight. They were incubated with biotin-conjugated goat anti-rabbit Ig (Dako), for 30 min at RT, followed by the addition of peroxidase conjugated streptavidin (Nichirei) for 5 min. Peroxidase activity was visualized by diaminobenzidine. The sections were counterstained with Mayer’s Hematoxylin (MUTO), dehydrated, and then mounted with Malinol (MUTO).

The hypothalami from young and aged male and female mice were rapidly removed while being cooled by ice 6 h after stress exposure and were perfusion-fixed with formaldehyde. For the first immunohistochemistry analysis, tissue sections were de-paraffinized with xylene and rehydrated through an ethanol series and Tris-buffered saline. Antigen retrieval was performed by microwave treatment, with citrate buffer, pH 6.0. Endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol for 30 min, followed by incubation with Protein Block (Genostaff, Tokyo, Japan) and an avidin/biotin blocking kit (Vector). The sections were incubated with anti-ERα rabbit polyclonal antibody (Santa Cruz) at 4°C overnight. They were incubated with biotin-conjugated goat anti-rabbit Ig (Dako, Tokyo, Japan) for 30 min at RT, followed by the addition of peroxidaseconjugated streptavidin (Nichirei, Tokyo, Japan) for 5 min. Peroxidase activity was visualized by diaminobenzidine and then each section was washed with PBS.