Sm2000r freezing microtome

Brain samples were fixed in 10% formalin for 90–120 min at room temperature, then transferred into 15 and 30% sucrose solutions for 24 h at 4∘C prior to being serially sectioned at 50 μm on a SM2000 R freezing microtome (Leica Microsystems, Concord, ON, Canada). Tissue sections were then processed for free-floating immunohistochemistry to label cells expressing Sox2, PCNA, or DCX. In brief, sections were incubated in 0.9% H₂O₂ and blocked overnight at room temperature in PBS containing 0.1% Triton and 3% NS. Sections were then incubated in PBS containing 3% NS and one of the primary antibodies described above (1:500 Sox2; 1:1000 PCNA; 1:1000 DCX) for 48–96 h at 4∘C. After washing, tissues were incubated in PBS containing 3% NS and secondary antibody at a concentration of 1:200, followed by incubation in ABC and development with DAB (Vector Laboratories, Burlingame, CA, USA). Sections were then mounted on slides, dehydrated, and coverslipped with Permount. Contrast and exposure have been modified on some of the presented images to improve visibility of cellular structures.

Mice (n = 6 per genotype/age/sex) were euthanized at 4, 12, and 18 months via CO₂ inhalation and transcardially perfused with 1X phosphate buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, United States). These timepoints were selected to enable a standardized comparison of phenotyping data of the present systematic characterization of the 3xTg-AD mouse model and our previous systematic characterization of the 5xFAD mouse model. For all subsequent analyses, brains were removed with hemispheres separated along the midline. Brain halves were either drop-fixed in phosphate buffered 4% paraformaldehyde (Thermo Fisher Scientific, Waltham, MA, United States) at 4°C over a 24-h period for immunohistochemical staining or micro-dissected (cortex, hippocampus, midbrain) and flash frozen for biochemical analysis. Fixed half brains were coronally sliced at 40 μm using a Leica SM2000R freezing microtome. All brain hemispheres were processed and representative slices (between −2.78 mm posterior and −3.38 mm posterior to Bregma according to the Allen Mouse Brain Atlas, Reference Atlas version 1, 2008) containing hippocampal and cortical regions from each mouse were used for histological staining and stored in 4°C in cryoprotectant.

All animal experiments were performed according to animal protocols approved by the Institutional Animal Care and Use Committee at UCI, an American Association for Accreditation of Laboratory Animal Care (AAALAC)‐accredited institution. Four‐ and 12‐month‐old mice were euthanized using CO₂ inhalation, then transcardially perfused with 1x phosphate‐buffered saline (PBS). The brains were collected and divided into hemispheres; one was flash frozen for biochemical analysis or RNA sequencing (RNA‐seq), and the other was drop‐fixed in 4% paraformaldehyde (PFA; Thermo Fisher Scientific, Waltham, MA, USA) for immunohistochemical analysis. Fixed brains were cryopreserved in PBS + 0.05% sodium azide + 30% sucrose, and a Leica SM2000R freezing microtome was used to generate 40‐μm‐thick coronal brain slices. The coronal brain slices were collected between −2.78 mm posterior and −3.38 mm posterior to Bregma according to the Allen Mouse Brain Atlas (Reference atlas version 1, 2008). Brain sections were stored in 30% glycerol + 30% ethyl glycol in 1x PBS at −20°C until histological evaluation.

All rodent experiments were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, Irvine. 7-month-old wild-type (WT) or 5xFAD mice were intraperitoneally (IP) injected with 55 mg/kg G4 PAMAM hydroxyl dendrimers conjugated to a Cy5 fluorophore followed by euthanasia 48 h post injection. For time course D-Cy5 experiments, 7–9-month-old mice were treated as above, but euthanized either 48 h, 15 days, or 21 days post injection. For D-45113 experiments, 4 month and 11-month-old mice were IP injected with 200 mg/kg of D-45113 twice per week for four weeks. At the end of treatments, mice were euthanized via CO₂ inhalation and transcardially perfused with 1X phosphate buffered saline (PBS). For all studies, brains were removed, and hemispheres separated along the midline. Brain halves were either flash frozen for subsequent biochemical analysis, or drop-fixed in 4% Paraformaldehyde (PFA; Thermo Fisher Scientific, Waltham, USA) for subsequent immunohistochemical analysis. Half brains collected into 4% PFA for 48 h and then transferred to a 30% sucrose solution with 0.02% sodium azide for another 48–72 h at 4 C. Fixed half brains were sliced at 40 μm using a Leica SM2000 R freezing microtome.