Ab183947

Deparaffinized sections were boiled for 20 min in 10 mM citrate buffer (pH 6) (Genemed Biotechnologies) for antigen retrieval, rinsed in distilled water, and then blocked with 3% normal goat serum (Vector Laboratories) in PBS for 1 h at room temperature. The sections were incubated with primary antibodies in PBS or Can Get Signal immunostain solution (Toyobo, Osaka, Japan) for 1 h at room temperature. The primary antibodies were as follows: rabbit anti-GFAP, rabbit anti-Iba1, rabbit anti-NeuN, and rabbit anti-olfactory marker protein (OMP) (#ab183947, 1:1,000; Abcam, Cambridge, UK). After washing in PBS, the sections were incubated with Alexa Fluor 488- or 594-conjugated goat anti-rabbit IgG antibodies (#A31627 and #A-11012, 1:1,000; Thermo Fisher Scientific, Waltham, MA, USA) in PBS or Can Get Signal immunostain solution for 1 h at room temperature, washed in PBS, and mounted using VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories). All images were captured and analyzed using the ECLIPSE Ti confocal microscope (Nikon Instruments) with NIS-Elements AR imaging software version 4.00.06 (Nikon Instruments). The number of OMP-positive cells was measured in all bilateral subareas, and their mean values were calculated for each animal.

The process of immunofluorescence analysis was performed as described by our previous studies (Yin et al. 2013a (link)). Briefly, brain slices were permeabilized with 0.3% Triton-X 100 and 5% BSA in PBS and incubated with primary antibodies at 4 °C overnight. The brain slices were not treated with Triton-x 100 when staining with anti-GAD67 antibodies. After washing with PBS for three times, samples were incubated with Alexa Fluor-488 or -405 secondary antibodies (1:1000, Invitrogen) for 1 h at room temperature. Samples were mounted with Vectashield mounting medium (Vector) and images were taken by Leica TCS SP8 confocal microscope. The following primary antibodies were used: rabbit anti-Drd2 (1:200, Millipore, AB5084P), rabbit anti-NeuN (1:500, Abcam, ab177487), mouse anti-PV (1:500, Sigma, P3088), mouse anti-GAD67 (1:300, Millipore, MAB5406), mouse anti-TBX21 (1:100, Abcam, ab91109), and rabbit anti-OMP (1:200, Abcam, ab183947).