Immunohistochemistry and confocal microscopy
Mice were deeply anaesthetized with pentobarbital (150 mg/kg i.p.) followed by transcardial perfusion-fixation with phosphate-buffered saline (PBS, pH 7.4, 20 ml) and then freshly-prepared ice-cold paraformaldehyde (PFA, 4% in 0.4 M sodium phosphate buffer, 50 ml). Brains were extracted and placed in 4% PFA for post-fixation for 5 h, and transferred into 30% sucrose solution for 48 h, before embedding in cryoprotective medium (Tissue-TEK OCT Compound), freezing on powdered dry ice and storing at -80 °C. Using a cryostat (Leica) at -18°C, brains were sectioned coronally at 40 µm. A mouse brain atlas was used to identify sections at the appropriate bregma levels. For c-Fos immunofluorescence, 5 series of sections including BA were collected from -1.0 to -2.6 mm, 1 section per 200 µm per series. The sections were stored in cryoprotective solution (glycerine and ethylene glycol in 0.2 M phosphate buffer; Sigma-Aldrich) at -20°C until further processing. For c-Fos immunofluorescence, sections were transferred to a 24-well plate (2 sections/well) and washed 3 x 20 s in Tris-Triton buffer (pH 7.4) on a shaker. Sections were blocked in 2% goat or donkey serum (Sigma-Aldrich) in 0.2% Triton X-100 in Tris-Triton for 60 min at RT on a shaker, and incubated with 1:1000 rabbit anti-c-Fos 9F6 (Cell Signaling Technology Cat# 2250, RRID:AB_2247211)) in blocking solution for 18h at 4°C on a shaker. Sections were washed in Tris-Triton for 3 x 20 s at RT and incubated with secondary antibody in blocking solution for 120 min at RT on a shaker: depending on the CTB conjugated fluorophore, either 1:500 goat anti-rabbit cyanine5 (A10523, ThermoFisher Scientific, RRID:AB_2534032)) or 1:500 donkey anti-rabbit Alexa Fluor 488 (A-21206, ThermoFisher Scientific, RRID:AB_2535792).-. The sections were washed 3 x 20 s in Tris-Triton, transferred to 1x PBS, and mounted on microscope slides (SuperFrost, Epredia). Fluoroshield with DAPI (F6057, Sigma-Aldrich) was added, and the sections cover-slipped for microscopy.
Section images were acquired using an inverted confocal laser scanning microscope (Leica SP8) fitted with an HC PL APO CS2 20x NA 0.75 multi-immersion objective in standard mode. The pinhole was set to 1 AU (Airy units), pixel size to 1.14 x 1.14 µm2, and z-stack step size to 2 µm. Following the acquisition of the z-stack images, the area of interest per hemisphere was defined and these were analyzed using a macro custom-written in Fiji foridentification and quantification of CTB, c-Fos and DAPI, with the experimenter blinded with respect to mouse ID and group. Areas that were CTB+ or c-Fos+ were automatically filtered for size, with the range 10-300 pixels used to define cell bodies. CTB+ or c-Fos+ areas were also automatically filtered with respect to the threshold for the gray value that differentiated signal from background: this was established empirically for each marker; for CTB one threshold was used for all images of each mouse and adjusted mildly between mice, and for c-Fos one threshold was used across all images. Visual inspection of the binary mask output confirmed that automated counting yielded a representative quantification of the confocal micrographs. The numbers and densities of CTB+, c-Fos+ and CTB+/c-Fos+ cells were determined, and the mean numbers/densities per hemisphere were calculated for each subject.