Freezing microtome

Mice were anesthetized and perfused with phosphate-buffered saline (PBS) and paraformaldehyde (PFA) in phosphate buffer (0.2 M). After dehydration, the brains were sectioned at an equal thickness of 40 μm using a freezing microtome (Leica Biosystems, Nussloch, Germany). Sections were cleaned using PBS, activated with 1% hydrogen peroxide (H₂O₂) for 15 min, and blocked in a solution containing 0.3% bovine serum albumin (BSA) and 3% Triton X-100 for 1 h. After blocking, the sections were activated using the following primary antibodies: anti-tyrosine hydroxylase (TH; rabbit, 1:1,000 for ST and 1:3,000 for SNpc, Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-14007) and c-Fos antibody (rabbit, 1:500, Santa Cruz Biotechnology; sc-253). After several rinses, the sections were incubated for 1 h in PBS containing a biotinylated anti-rabbit secondary antibody (Vector Laboratories Inc., Burlingame, CA, USA; BA-1000). Immunoreactions were visible after incubation for 1 h at room temperature in PBS containing avidin-biotinylated peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories Inc.) and in 3-3-diamiobenzidine tetrahydrochloride (Vector Laboratories Inc.; SK-4100). After several rinses, the sections were mounted on slides, dehydrated, and covered with coverslips. Histological images were obtained (Olympus Japan Co., Tokyo, Japan; BX53).

The animals were deeply anesthetized using vaporized isofluorane and euthanized by rapid decapitation. Brains were rapidly dissected, flash-frozen in 2-methylbutane at −30 °C, and then sectioned coronally at 200 μm on a freezing microtome (Leica Biosystems, Lincolnshire, IL, USA). The paraventricular nucleus (PVN) was microdissected using a 0.75-mm Palkovit’s brain punch tool (Stoelting, Inc., Wood Dale, IL, USA) at −1.49 to −2.12 relative to bregma, as defined by The Rat Brain in Stereotaxic Coordinates [43 ]. Frozen PVN microdissections were transferred to a microcentrifuge tube and stored at −80 °C.

The rats were sacrificed immediately following cystometric determination of the contraction pressure and time in the bladder. Briefly, the animals were anesthetized with Zoletil 50^® (10 mg/kg, i.p.; Vibac Laboratories), and were transcardially perfused with 50 µm phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 100 mm sodium phosphate buffer at pH 7.4. The brain was removed, postfixed in the same fixative overnight and transferred to a 30% sucrose solution for cryoprotection. Subsequently, 40 µm serial coronal sections were produced with a freezing microtome (Leica Biosystems Nussloch GmbH, Nussloch, Germany). The PMC was defined as the region spanning Bregma −9.68 to −9.80 mm, the vlPAG as the region spanning Bregma −7.64 to −8.00 mm, the MPA as the region spanning Bregma −0.26 to 0.80 mm and the spinal cord as the L4–L5 region. An average of 10 sections were collected from each rat for each region and were used for immunohistochemical analysis.

Immunofluorescence staining was performed for macrophage biomarker F4/80 and neutrophil membranous biomarker Ly6G. Removed kidney tissues were frozen immediately with liquid nitrogen and then were cut into 2 μm thick frozen sections with the help of freezing microtome (Leica Biosystems, Germany) by skilled technician. Endogenous peroxidase was blocked for 20 min in 3% hydrogen peroxide, and PBS was used to rinse the samples. After blocking with 5% BSA, the slides were incubated at 4°C with a rabbit anti-mouse Ly6G (dilution 1 : 100; Abcam, USA) and F4/80 antibody (1 : 50, Invitrogen, Life Technologies, USA) overnight. The anti-rabbit fluorescein-conjugate second antibody (Invitrogen, Thermo Fisher Scientific, USA) was used to detect and amplify the primary signals by light emission. The pictures were observed and evaluated under fluorescence microscopy (Observer A1 inverted microscope, ZEISS, Germany) with 400x actual magnification. Two observers evaluated the slides, who were unaware of the slides classification. F4/80-positive and Ly6G-positive cell number in each section were calculated by counting positively stained cells in 10 fields per slide at 400x magnification.

Male and female WT and APP/PS1 mice were sacrificed 1-month post-surgery, while male and female CX3CR1^GFP/+ → WT mice were divided into 3 subgroups that were sacrificed 3 days, 1 week and 1 month post-surgery. Male APP/PS1/CX3CR1^GFP/+ mice were sacrificed 1 week post-surgery. Mice were deeply anesthetized with a solution containing ketamine (90 mg/ml) and xylazine (10 mg/ml) and sacrificed via intracardiac perfusion with 0.9% NaCl. For immunohistochemical analysis, the brains were retrieved, post-fixed in 4% paraformaldehyde (PFA) for 24 hours and transferred into 20% sucrose containing 4% PFA for a minimum of 24 hours. Brains were then cut into 25 μm coronal sections using a freezing microtome (Leica Biosystems, ON, Canada), and serial sections were collected in 12 well-plates in an antifreeze solution (30% glycerol, 30% ethylene glycol in 0,9% NaCl, phosphate buffer (PB)) and kept at -20°C for further use. For Western blot analysis, the brains were removed and dissected to separate the hippocampus and cortex from each hemisphere that were snap-frozen on dry ice and kept at -80°C until further use. Each region was then homogenized separately in a NP40 lysis buffer containing 1% of phosphatase inhibitors and 1% of protease inhibitors, sonicated, and then stored in -80°C until further use.