Euthasol® (39 mg pentobarbital sodium, 5 mg phenytoin sodium, Virbac, USA) was subcutaneously injected to anesthetize mice. When mice were in deep anesthesia, the heart was exposed, and a 25G needle was inserted into the left ventricle. Mice were perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. Then, the intact brain and lumbar spinal cord were collected and fixed in 4% paraformaldehyde overnight at 4 °C, followed by dehydration in 30% sucrose solution until the tissue sank to the bottom of the bottles. After fixation and dehydration, the whole brain and spinal cord were dissected and embedded in OCT compound, and 35-μm slices were cut with a cryostat (CM1850 UV, Leica, Germany). The sections containing the brain areas, i.e., the prelimbic cortex (bregma 1.94 mm) of prefrontal cortex (PFC), anterior cingulate cortex (ACC, bregma 0.74 mm), the hippocampus (bregma − 2.06 mm) encompassing the dentate gyrus (DG) and CA3, and the L4-L6 spinal cord, were collected according to the spatial coordinates of the coronal plane in the mouse brain atlas [36 ] and mouse spinal cord atlas [37 ], respectively.
For immunofluorescence staining, brain slices were (1) washed in PBS containing 0.3% Triton and blocked for 2 h at room temperature with 1% bovine serum albumin and 2% donkey or goat serum; (2) incubated overnight at 4 °C with primary antibodies: rabbit anti-Iba-1 (1:500, 019-19741, Fujifilm Wako, Japan), rabbit anti-tyrosine hydroxylase (TH, 1:500, ab112, Abcam, UK), mouse anti-norepinephrine transporter (NET, 1:500, ab211463, Abcam), and mouse anti-Aβ42 (1:500, 05-831-I, Millipore, USA); (3) washed with PBS containing 0.3% Triton and incubated for 2 h at room temperature with anti-rabbit Alexa Fluor® 488 (111-545-003, Jackson ImmunoResearch, USA; or ab150073, Abcam) or anti-mouse Cy3 (ab97035, Abcam) secondary antibodies; and (4) washed in PBS containing 0.3% Triton before being mounted with mounting medium containing Fluoroshield (ab104139, Abcam). Immunofluorescence pictures were taken with a fluorescence microscope (80i, Nikon, Japan) and CoolSNAP DYNO CCD (Photometrics, Canada).
To quantify Iba-1, TH, NET, and Aβ42 expression, the brain and spinal cord images (magnification = × 100) were outlined with the size-standardized regions of interest (ROIs) by the Image J software (v.1.52a, NIH, USA), and the percentage of area with fluorescence was quantified using this program. In particular, the threshold was set and standardized across images to maximize true protein expression signal for quantification; then, the total pixel number of target protein were recorded, and the percentage was calculated by dividing the pixel number with the total unfiltered pixel number in the ROI. TH+ neurons in the LC [38 (link)] (bregma − 5.4 mm [36 ]) were captured under the same magnification and counted manually [39 (link)]. Three sections for each brain and spinal cord region per immunostaining marker were averaged and analyzed. To evaluate non-specific staining, incubation of sections in primary or secondary antibody were conducted for each round of staining, and the resulting images confirmed that the primary and the secondary antibodies did not cause nonspecific staining.
For immunofluorescence staining, brain slices were (1) washed in PBS containing 0.3% Triton and blocked for 2 h at room temperature with 1% bovine serum albumin and 2% donkey or goat serum; (2) incubated overnight at 4 °C with primary antibodies: rabbit anti-Iba-1 (1:500, 019-19741, Fujifilm Wako, Japan), rabbit anti-tyrosine hydroxylase (TH, 1:500, ab112, Abcam, UK), mouse anti-norepinephrine transporter (NET, 1:500, ab211463, Abcam), and mouse anti-Aβ42 (1:500, 05-831-I, Millipore, USA); (3) washed with PBS containing 0.3% Triton and incubated for 2 h at room temperature with anti-rabbit Alexa Fluor® 488 (111-545-003, Jackson ImmunoResearch, USA; or ab150073, Abcam) or anti-mouse Cy3 (ab97035, Abcam) secondary antibodies; and (4) washed in PBS containing 0.3% Triton before being mounted with mounting medium containing Fluoroshield (ab104139, Abcam). Immunofluorescence pictures were taken with a fluorescence microscope (80i, Nikon, Japan) and CoolSNAP DYNO CCD (Photometrics, Canada).
To quantify Iba-1, TH, NET, and Aβ42 expression, the brain and spinal cord images (magnification = × 100) were outlined with the size-standardized regions of interest (ROIs) by the Image J software (v.1.52a, NIH, USA), and the percentage of area with fluorescence was quantified using this program. In particular, the threshold was set and standardized across images to maximize true protein expression signal for quantification; then, the total pixel number of target protein were recorded, and the percentage was calculated by dividing the pixel number with the total unfiltered pixel number in the ROI. TH+ neurons in the LC [38 (link)] (bregma − 5.4 mm [36 ]) were captured under the same magnification and counted manually [39 (link)]. Three sections for each brain and spinal cord region per immunostaining marker were averaged and analyzed. To evaluate non-specific staining, incubation of sections in primary or secondary antibody were conducted for each round of staining, and the resulting images confirmed that the primary and the secondary antibodies did not cause nonspecific staining.
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