Alexa fluor 594 goat anti rabbit igg

For double immunofluorescent staining of Bax and Bcl-2, the sections were first covered with 3% BSA at 37°C for 30 min and incubated with a rabbit polyclonal antibody against rat Bax (1 : 50, Abcam, USA) at 4°C for overnight in a wet box. After shaking three times in PBS (pH 7.4) on a decolorized shaker for 5 min each, sections were subsequently incubated with Alexa Fluor 594 goat anti-rabbit IgG (1 : 200 dilution, Abcam, USA) at 37°C for 30 min. After rinsing thoroughly in PBS (pH 7.4), the same procedure was repeated with a rabbit polyclonal antibody against rat Bcl-2 (1 : 100, Cell Signaling Technology, USA) and an Alexa Fluor 488 goat anti-rabbit (1 : 200 dilution, Abcam, USA). For dual staining of Akt and NF-κB, the sections were stained with a rabbit polyclonal antibody against rat Akt (1 : 300, Abcam, USA), a rabbit monoclonal antibody against rat NF-κB p50 (1 : 100, Abcam, USA), an Alexa Fluor 594 goat anti-rabbit IgG (1 : 200 dilution, Abcam, USA), and an Alexa Fluor 488 goat anti-rabbit (1 : 200 dilution, Abcam, USA). Sections were visualized using UV excitation wavelength 330–380 nm, emission wavelength 420 nm; FITC green excitation wavelength 465–495 nm, emission wavelength 515–555 nm; and CY3 red light excitation wavelength 510–560 nm, emission wavelength 590 nm with the Nikon Eclipse Ti-SR (Nikon, Tokyo, Japan) and Nikon DS-U3 (Nikon, Tokyo, Japan).

B6.Cg-Tg(Mc4r-MAPT/Sapphire)21Rck/J male mice were perfused transcardially via a 23-gauge needle placed in the left ventricle with 100 ml of 0.1 M heparinized PBS, pH 7.4, followed by 100 ml of 4% paraformaldehyde in PBS, and the fixed brains were cryoprotected in 30% sucrose for 48 h at 4C. Coronal hypothalamic sections of 30 μm thickness were prepared on a freezing microtome. Free floating sections were incubated for 15 min in 1% hydrogen peroxide, washed 2 times in PBS, blocked 2 h in 0.3% Triton X-100 and 5% NGS in PBS, before being incubated overnight at 4 °C in rabbit anti-proopiomelanocortin precursor (1:200, Phoenix Pharmaceuticals) in 0.3% Triton X-100 and 5% NGS. Sections were then washed and incubated for 2 h with Alexa Fluor 594 goat anti-rabbit IgG. Sections were washed and incubated overnight at 4 °C in chicken anti-GFP (1:500, Abcam). Sections were washed and incubated in Alex Fluor 488 goat anti-chicken IgG for 2 h. Sections were floated onto superfrost Plus microscope slides (Fisher), and coverslipped with Vectashield (Vector). Tissue sections were digitized using a Zeiss Axioplan fluorescent microscope, and areas of interest were outlined based on cellular morphology.

A triple-staining procedure was implemented for BrdU/DCX/NeuN to allow analyzation through immunofluorescence. The brain sections were incubated overnight at 4°C with the following primary antibodies: rat anti-BrdU (1:200, ab6326, Abcam), mouse anti-NeuN (1:500, ab104224, Abcam) and rabbit anti-DCX (1:500, ab18723, Abcam). The sections were washed in PBS and incubated for 4 h with fluorescent secondary antibodies: Alexa Fluor 488 goat anti-rat IgG to reveal immunoreactivity of BrdU, Alexa Fluor 405 donkey anti-mouse IgG to reveal immunoreactivity of NeuN, and Alexa Fluor 594 goat anti-rabbit IgG to reveal immunoreactivity of DCX, respectively (1:400 for all three antibodies, Abcam). Every sixth coronal section from the adult dentate gyrus was used to assess BrdU+/NeuN+/DCX- and BrdU+/NeuN+/DCX+ positive cells in both hemispheres (10 sections per animal, 3 mice per group). BrdU+/NeuN+/DCX- and BrdU+/NeuN+/DCX+ positive cells were observed and counted under an Olympus BX63 microscope fitted with a ×10 eyepiece lens with a ×40 objective, and their percent distribution in the BrdU positive cells was calculated.