Mouse anti neun

For detection of BrdU-labeled nuclei, sections were incubated with 1N HCl at 45°C for 30 min, and then neutralized in 0.1 M borate buffer (PH 8.5) for 10 min. Sections were washed 3 times with PBS and then blocked using 10% calf serum for 2 h at room temperature. After blocking, sections were incubated overnight with mouse monoclonal anti-BrdU antibody (1:300; Biolegend) at 4°C. Co-labeling runs used rabbit anti-NeuN (1:500; Abcam) for the visualization of neurons. For immunofluoresence staining with Brdu, sections were incubated with primary antibodies as follows: rabbit anti-p75 (1:100; Ruiying Biological), mouse anti-NeuN (1:100; Santa Cruz Biotechnology) and mouse anti-GFAP (1:100; Santa Cruz Biotechnology). Sections were washed 3 times in PBS and incubated with appropriate fluorescence-conjugated secondary antibodies as follows: Alexa Fluor 555 donkey anti-mouse, Alexa Fluor 488 donkey anti-rabbit, and Alexa Fluor 405 Goat anti-rabbit IgG (1:500; Biolegend). Sections were mounted after washing with PBS and visualized with confocal microscopy.

The slices were rinsed with PBS for 30 min and permeabilized with 0.3% Triton X-100 for 30 min at room temperature [37 ]. The slices were then rinsed with PBS for 15 min and blocked with 5% donkey serum at 37 °C for 30 min. Subsequently, each coronal slice was incubated with primary antibodies at 4 °C overnight. The primary antibodies used are as follows: rabbit anti-p-IRE1α (1:50, Abcam), mouse anti-NeuN (1:200, Santa Cruz Biotechnology), mouse anti-GFAP (1:100, Santa Cruze Biotechnology), and mouse anti-Iba-1(1: 200, Wako). Slices were rinsed in PBS for 15 min and then incubated with appropriate fluorescence-conjugated secondary antibodies at 37 °C for 1 h. After being rinsed in PBS for 15 min, the slices were covered with Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories Inc.). Images were then visualized under a fluorescence microscope (Leica DMi8, Leica Microsystems).