Mice were anaesthetized with 200 mg/mL sodium pentobarbital and intracardially perfused with PBS, pH 7.4, and 4% paraformaldehyde. The brains were post-fixed and cut into six series of coronal sections (40 µm) using a vibratome (Leica VT1000S, Leica Biosystems). The following primary antibodies were used to identify microglia and immature neurons: rabbit anti-Iba1 (1:500; Wako, ref: 019-19741) and goat anti-DCX (1:200; Santa Cruz Biotechnology, ref: sc-8066). Then, we used the corresponding biotinylated secondary antibodies: anti-rabbit (1:1000; Dako, ref: E0432) and anti-goat (1:1000; Dako, ref: E0466) [36 (link)].
To identify new neurons, control and stress mice received three intraperitoneal administrations of BrdU (50 mg/kg, Sigma–Aldrich, Madrid, Spain) separated by 3 h just after acute stress treatment. Double immunofluorescence labelling was carried out combining the rat anti-BrdU antibody (1:1000; Accurate Chemical, ref: OBT0030) and the rabbit anti-DCX antibody (1:600; Abcam, ref: ab18723), followed by the corresponding secondary antibodies of Alexa Fluor 488 anti-rat (1:1000; Invitrogen, ref: A21208) and Alexa Fluor 568 anti-rabbit (1:1000; Invitrogen, ref: A10042). DAPI was used as a nuclear contrast stain [36 (link),37 (link)] (seeSupplementary Information for details).
To identify new neurons, control and stress mice received three intraperitoneal administrations of BrdU (50 mg/kg, Sigma–Aldrich, Madrid, Spain) separated by 3 h just after acute stress treatment. Double immunofluorescence labelling was carried out combining the rat anti-BrdU antibody (1:1000; Accurate Chemical, ref: OBT0030) and the rabbit anti-DCX antibody (1:600; Abcam, ref: ab18723), followed by the corresponding secondary antibodies of Alexa Fluor 488 anti-rat (1:1000; Invitrogen, ref: A21208) and Alexa Fluor 568 anti-rabbit (1:1000; Invitrogen, ref: A10042). DAPI was used as a nuclear contrast stain [36 (link),37 (link)] (see
Free full text: Click here
