Neuronal CCL2 expression drives inflammatory monocyte infiltration into the brain during acute virus infection

Charles L Howe; Reghann G. LaFrance-Corey; Emma N Goddery; Kanish Mirchia

Terminally anesthetized mice (isoflurane overdose) were perfused with 50 mL of 4% paraformaldehyde (PFA) via intracardiac puncture and tissues were postfixed in 4% PFA for 24 hours. Brain tissue was macrosectioned using a brain matrix, making cuts through the optic chiasm and infundibulum. The resulting tissue block containing the dorsal hippocampus was embedded in 4% agarose gel and sectioned at 70 µm thickness by vibratome. Free floating sections were blocked in PBS containing 1% BSA, 10% normal donkey serum, 1% FBS, and 0.1% Triton-X 100 for 1 hour, incubated overnight at 4°C with primary antibody (anti-MCP-1/CCL2: Cell Sciences, CPM001, 1:400 in block), incubated with secondary antibody for 1 hour (donkey anti-rabbit Cy3: Jackson ImmunoResearch, 711-166-152, 1:1000 in block), washed, and mounted in DAPI-containing mountant on charged slides. CCL2 immunoreactivity was imaged with the Zeiss AxioObserver.Z1 and ApoTome.2 structured illumination system (Carl Zeiss Microscopy GmbH, Jena, Germany) using a 20x objective (LD Plan-Neofluar 20x/0.4 Korr Ph 2 M27, 0.55 NA), 538-562 nm bandpass excitation, 570-640 nm bandpass emission, 7 µm optical thickness, and 300 msec exposure time. For detection of the mCherry fluorophore in CCL2:RFP animals, free floating 70 µm sections were mounted on charged slides in DAPIcontaining mountant. Fluorescence images were captured with a laser scanning confocal microscope (LSM780, Carl Zeiss Microscopy GmbH, Jena, Germany) using a 40x objective (C-Apochromat 40x/1.20 W Korr FCS M27, 1.2 NA) with water as the refractive medium. Validation of mCherry-specific emission and exclusion of autofluorescence was obtained with spectral imaging using a lambda scan at 488 nm, 561 nm, and 594 nm excitation and 8 nm-stepped emission spectra. 40 µm z-stacks were acquired with a step thickness of 2 µm, pixel dwell time of 0.39 µs, and pinhole equivalent to 1 airy unit across all samples. Uncompressed TIFF-images were exported from Zen software (Zen Black 2012 64-bit, Carl Zeiss Microscopy GmbH, Jena, Germany) and post-processed in ImageJ (ImageJ v1.50b, Wayne Rasband, National Institutes of Health, USA) and Photoshop (Adobe Photoshop CC, 2014 Release, 64-bit). Levels were normalized, when appropriate, equally across images and groups; gamma values were not changed.