3 3 diaminobenzidine dab

Paraffin sections of the hippocampus and hypothalamus from different groups were used for ezrin immunohistochemical staining. The expression of ezrin proteins was detected using avidin–biotin complex (ABC) method (Ramprasad et al. 1996 (link)). Deparaffinized and rehydrated sections were rinsed in distilled water for 5 min, washed in PBST for 10 min, and incubated with 10% normal goat serum for 15 min to reduce non-specific background staining. Then, the sections were incubated with anti-rabbit ezrin (Dako, 1:100) for 1–2 h at room temperature. The sections after 5 baths in PBST were incubated with biotinylated goat anti-rabbit immunoglobulin (Nichirei, Tokyo, Japan) for 1 h at ambient temperature. The sections were incubated with 3–3’-diaminobenzidine (DAB, Wako pure chemical industries, Ltd) for 7–9 min in dark at room temperature. Negative control sections were done by substituting ezrin primary antibodies by normal serum of rabbit. The prepared sections were examined by means of a research microscope and analyzed for the positive ezrin-immunostaining using the ImageJ analyzer (version 5.1).

Tissues were fixed in 4% paraformaldehyde (Wako Pure Chemical) for 24 h and stored in 10% sucrose in PBS at 4°C. Fixed tissues were dehydrated with 70% ethanol, acetone and 100% ethanol, transferred into xylene (10 minutes × 3 times), and paraffin (30 minutes × 3 times, at 60°C), and then embedded in paraffin. Paraffin-embedded tissue blocks were cut into 10 μm thick sections using a sliding microtome (Nippon Optical Works, Tokyo, Japan), and extended on MAS-coated glass slides (Matsunami Glass, Kishiwada, Japan). After drying at 60°C for 24 h, the sections were deparaffinized with xylene (10 minutes × 3 times) and ethanol (100%, 90%, 80% and 70%), washed with PBS, and then stained with antibodies against phosphorylated tau at Ser/Thr 202/205 (AT8, 1:100 dilution; Thermo Fisher Scientific) or neuron (NeuN, 1:100 dilution; Millipore), using M. O. M Immunodetection Peroxidase kit (Vector Laboratories, Burlingame, CA, USA). 3,3’-diaminobenzidine (DAB, Wako Pure Chemical) at a concentration of 0.5 mg/ml in PBS with 0.005% hydrogen peroxide was used as chromogen. The sections were dehydrated with ethanol (70%, 80%, 90%, and 100%) and xylene, and then mounted with mounting medium (Daido Sangyo, Tokyo, Japan). The sections were observed using a microscope BZ-8100 (Keyence, Osaka, Japan)