Mice
Wild-type (WT), Mac-1
−/− [
60], CX3CR1
GFP/CCR2
RFP (R/G) [
31,
57],
Pdgfra H2B-
eGFP (
Pdgfra
GFP/+) [
25], and the B6.129S7-Gt(ROSA)26Sor/J (ROSA) [
22] mice were all in the C57BL/6J background and used at ~8–13 weeks (20–22 g). WT, R/G, and ROSA mice were purchased from the Jackson Laboratory. Mac-1
−/− mice were kindly provided by Dr. Christie M. Ballantyne, Baylor College of Medicine (Houston, TX, USA), and the
Pdgfra
GFP/+ mice by Dr. Philippe M. Soriano, Icahn School of Medicine at Mount Sinai (New York, NY, USA). Mac-1
−/− ROSA mice were generated by crossing ROSA with Mac-1
−/− mice. The bitransgenic R/G mice are heterozygous for CX3CR1
GFP/CCR2
RFP expressing GFP in microglia and macrophages under the control of the CX3CR1 promoter and RFP in monocytes and macrophages under the control of the CCR2 promoter. These bitransgenic knock-in mice are used as heterozygous mice to avoid deficiency of CCR2 or CX3CR1 gene expression. The
Pdgfra
GFP/+ express a nuclear GFP signal in cells, where the
Pdgfra promoter is/has been active. All mice were housed in a pathogen-free facility and all procedures were performed in accordance with the local welfare legislation and approved by the Institutional Animal Care and Use Committees at the University of Michigan, University of Maryland, Emory University and Karolinska Institutet.
Immunostaining and confocal microscopy
Tissue preparation for sectioning and immunostaining was conducted using standard protocols. In short, mice were anesthetized with isoflurane and then transcardially perfused with phosphate-buffered saline (PBS) followed by perfusion with 4% paraformaldehyde (PFA) in PBS. The brains were removed, post-fixed in 4% PFA 1 h at room temperature (RT), and submerged in 30% sucrose. The brains were thereafter cryopreserved in OCT and sectioned in a cryostat. Alternatively, the brains were sectioned using a vibratome. Following immunofluorescent staining, the sections were mounted using ProLong Gold Antifade reagent (P36930, Life Technologies, Molecular Probes, Grand Island, NY, USA). If not stated otherwise, images were acquired at RT with a Zeiss LSM700 confocal microscope and the ZEN 2009 software (Carl Zeiss Microimaging GmbH, Jena, Germany). The images shown are representative of the respective staining and were processed and analyzed using the Volocity 3D image analysis software (PerkinElmer, Waltham, MA, USA), Photoshop CS5 (Adobe, San Jose, CA, USA), or ImageJ64 (National Institutes of Health, Bethesda, MD, USA).
Brains from adult naïve WT mice were used for co-localization of CD11b, LRP1, NG2, GFAP, and PDGFRα in the neurovascular unit. Vibratome sections (50 μm sections) were stained free-floating in 24-well plates. The sections were permeabilized with 1% BSA in 0.5% TritonX-100/PBS over night at 4 °C followed by incubation with primary antibodies (1:200) in blocking solution 0.5% BSA in 0.25% TritonX-100/PBS over night at 4 °C. The specific primary antibodies used were: rat anti-CD11b (550282, BD Biosciences, Franklin Lakes, NJ, USA), rabbit anti-LRP1 (#Rab2629, made In House Prof. DK Strickland), rabbit anti-NG2 (AB5320, Merck/Millipore), rabbit anti-GFAP (Z0334, Agilent Technologies/Dako), and goat anti-PDGFRα (AF1062; R&D Systems, Minneapolis, MN, USA). Goat or rat anti-CD31 antibodies were used to visualize endothelial cells (AF3628, R&D Systems, UK; and 553370, BD Biosciences, respectively). After thorough wash, the sections were incubated with appropriate Alexa Flour®-conjugated secondary antibodies and Alexa Flour®-conjugated streptavidin (Life Technologies, Molecular Probes) in blocking solution 0.5% BSA in 0.25% TritonX-100/PBS over night at 4 °C, then washed and mounted.
For detection of reactive microglia and in vivo phosphorylation of PDGFRα following ischemic stroke, MCAO was induced in WT, Mac-1−/−, and Pdgfra
GFP/+ mice. At 6 and 24 h after MCAO, the brains were harvested, cryopreserved in OCT, and sectioned. The sections were blocked in TNB buffer (TSA biotin system, NEL700A001KT, Perkin Elmer), followed by incubation over night at 4 °C with primary antibodies diluted in TNB buffer. The specific primary antibodies used were: rabbit anti-Iba1 (1:250, 019-19741, Wako Pure Chemical Industries Ltd., Osaka, Japan), rabbit anti-phospho-tyrosine (pY) 754 or -1018 specific PDGFRα (1:200, pY754 #2992 and pY1018 #4547; Cell Signaling, Danvers, MA, USA). Goat anti-CD31 or goat anti-podocalyxin antibodies were used to visualize endothelial cells (1:250; R&D Systems, UK). Appropriate Alexa Flour®-conjugated secondary antibodies (Life Technologies, Molecular Probes) were used to detect the bound primary antibodies.
Microglia activation was quantified using PFA perfusion-fixed brains. Iba1-stained brain sections (12 μm) from Mac-1−/− and WT mice (n = 3 per genotype and timepoint) were analyzed by two independent investigators blinded to the study group. The individual observations are based on analysis of five fields of view taken in the penumbra and contralateral cortex of each animal with the 20× objective using the Zeiss LSM700 confocal microscope. Cell body area was quantified using Volocity 3D and vessel association using the NIH Image J software. For quantification of PDGFRα activation in situ, unfixed brains from Mac-1−/− (n = 8) and WT (n = 6) mice subjected to 6 h of MCAO were used. Images were captured with a Nikon Eclipse TE800 upright microscope (Nikon, Tokyo, Japan). Quantification was performed using the NIH Image J software and the area of antibody immunoreactivity was determined from four to seven images per animal and normalized to WT control. n indicates the number of individual mice used in the study. The result from all the fields of view in a given animal was averaged to obtain the value for that individual. Individual values and group mean ± SEM are shown.
The specificity of the phospho-tyrosine specific PDGFRα antibodies was confirmed by Western blot analysis (Online Resource 4). Briefly, C6 glioma cells (CCL-107, ATCC) expressing PDGFRα, and PAE cells expressing PDGFRβ but not PDGFRα (PAE-β) [
16], were utilized. Cells were starved for 6 h in F12 medium containing 0.5% serum and thereafter acutely stimulated with 20 ng/ml active PDGF-CC (produced in house) or PDGF-DD (1159-SB; R&D Systems, Minneapolis, MN, USA) for 10 min at 37 °C to selectively induce PDGFRα and PDGFRβ receptor phosphorylation, respectively. Non-stimulated cells served as controls. Cells were lysed directly in SDS sample buffer and the proteins separated by SDS-PAGE and transferred to a nitrocellulose membrane. After blocking, membranes were incubated with the pY754 or pY1018 specific PDGFRα antibodies (1:500). Horseradish peroxidase tagged secondary antibodies were thereafter applied to the membranes and signals were detected using ECL Prime reagent kit (both GE Healthcare, Uppsala, Sweden). After stripping, membranes were reprobed with a general mouse anti-phospho-tyrosine antibody (1:1000, pY99 #sc7020; Santa Cruz Biotechnology, Dallas, TX, USA) or a mouse anti-phospho-tyrosine-751 PDGFRβ antibody (1:500, pTyr751 #3166; Cell Signaling, Danvers, MA, USA). All membranes were probed with a mouse anti-β-Actin antibody (1:1000, #ab6276; Abcam, Bristol, United Kingdom) as a control for equal loading.