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.