Phase 1: Animal Model Preparation and Tissue Collection — Weeks 1-2
Use aged (12-month-old) 5xFAD mice (The Jackson Laboratory #034840-JAX) and age-matched C57BL/6J controls (n=8 per group, power calculation for Cohen's d=1.2). Perform transcardial perfusion with PBS followed by 4% paraformaldehyde after deep anesthesia with ketamine/xylazine. Collect brain hemispheres: one for fresh-frozen sectioning (-80°C storage) and one for paraffin embedding. Prepare 20μm cryosections and 5μm paraffin sections using standard protocols. Include positive control tissues (kidney, bone) known to express high SPP1 levels.
...Phase 1: Animal Model Preparation and Tissue Collection — Weeks 1-2
Use aged (12-month-old) 5xFAD mice (The Jackson Laboratory #034840-JAX) and age-matched C57BL/6J controls (n=8 per group, power calculation for Cohen's d=1.2). Perform transcardial perfusion with PBS followed by 4% paraformaldehyde after deep anesthesia with ketamine/xylazine. Collect brain hemispheres: one for fresh-frozen sectioning (-80°C storage) and one for paraffin embedding. Prepare 20μm cryosections and 5μm paraffin sections using standard protocols. Include positive control tissues (kidney, bone) known to express high SPP1 levels.
Phase 2: SPP1 Expression Localization by Immunofluorescence — Week 3
Perform multiplex immunofluorescence on brain sections to identify SPP1-expressing cell types. Use primary antibodies: goat anti-SPP1 (R&D AF808, 1:200), rabbit anti-CD31 (Abcam ab28364, 1:300) for endothelial cells, mouse anti-PDGFRβ (Abcam ab32570, 1:250) for pericytes, rat anti-CD68 (Bio-Rad MCA1957, 1:400) for macrophages/microglia, and mouse anti-GFAP (Sigma G3893, 1:500) for astrocytes. Apply sequential staining protocol with appropriate blocking steps using 10% normal donkey serum. Use species-specific Alexa Fluor secondary antibodies (1:500) and DAPI nuclear counterstain. Image using confocal microscopy with 40x and 63x objectives, acquiring z-stacks (0.5μm steps) of perivascular regions identified by CD31+ vessels.
Phase 3: Quantitative Analysis of Cellular SPP1 Expression — Week 4
Perform systematic quantitative analysis of SPP1 expression in different cell populations. Use stereological sampling to analyze perivascular regions (within 50μm of CD31+ vessels) across hippocampus, cortex, and thalamus. Employ colocalization analysis using ImageJ with Coloc2 plugin to determine Manders' colocalization coefficients between SPP1 and cell-type markers. Quantify SPP1+ cell density (cells/mm²) for each cell type and calculate percentage of each cell type expressing SPP1. Include analysis of SPP1 fluorescence intensity using identical imaging parameters and exposure times across all samples.
Phase 4: Single-cell Resolution Analysis and Validation — Week 5
Perform high-resolution confocal imaging (100x oil objective) to confirm cellular identity of SPP1-expressing perivascular cells. Use orthogonal views and 3D reconstruction to verify colocalization. Validate findings using RNAscope fluorescent in situ hybridization (Advanced Cell Diagnostics) with probes for mouse Spp1 (Cat# 404611), Pdgfrb (Cat# 407131), and Cd68 (Cat# 316541). Process according to manufacturer's protocol with protease treatment and target retrieval. Quantify RNA signals using automated spot counting with minimum 3 spots per cell for positive scoring.
Phase 5: Western Blot Validation and Statistical Analysis — Week 6
Extract proteins from microdissected perivascular-enriched tissue using RIPA buffer with protease inhibitors. Perform Western blot using anti-SPP1 antibody (Abcam ab8448, 1:1000) with β-actin normalization (Sigma A2228, 1:5000). Use densitometry to quantify protein levels. Apply two-way ANOVA for statistical analysis with factors of genotype (5xFAD vs control) and brain region, followed by Sidak's multiple comparison test. Use unpaired t-tests for pairwise comparisons of cell-type specific SPP1 expression levels between genotypes.