Fig 2: TSPO expression in major cell types of the normal mouse brain. (A) Double immunofluorescence staining for TSPO (red) demonstrates punctate staining which colocalizes strongly with CD31+ vascular endothelial cells (green). This is observed in larger blood vessels, smaller arterioles and venules, as well as capillaries. Punctate staining is indicative of mitochondrial localization. (B) TSPO expression (red) and PDGFRß+ pericytes/smooth muscle cells (green) colocalize on larger blood vessel walls. (C) No clearly discernible TSPO expression (red) is observed in CD11b+ microglia (green). (D) TSPO expression (red) is not observed in GFAP+ astrocytes (green). Perivascular astrocytic endfeet and processes surround CD31+ endothelial cells of blood vessels, where TSPO strictly colocalizes. Scale bars = 20 µm, 10 µm for box insets.

Fig 3: Col2-creERmarks cells earlier than Osx-creER+ cells in the osteoblast lineage in fetal micea,b. Pregnant mice received 1mg tamoxifen at E11.5, and Col1-GFP; Col2-creER; R26RTomato mice were chased for 24 hours (a; E12.5) or 4 days (b; E15.5). Shown are distal halves of growth cartilages. Perichondrium is on top. Femur sections were stained for nuclei and CD31. Arrows: Tomato+ perichondrial cells, asterisks: Tomato+ chondrocytes, sharps: Tomato+ cells in primary ossification center. Green: EGFP, red: tdTomato, blue: Alexa633, gray: DAPI.c.d. Lineage-tracing of embryonic Col2-creER+ and Osx-creER+ cells was performed by injecting 1mg tamoxifen into pregnant mice at E11.5, and Col2-creER; R26RTomato (c) or Osx-creER; R26RTomato (d) mice were chased until P0. Shown are femur sections with the distal side on the left. Col2creER-E11.5 represents descendants of Col2-creER+ cells at E11.5. White indicates tdTomato.e,f. Pregnant mice received 1mg tamoxifen at E13.5, and Col2-creER; R26RTomato mice were chased for 24 hours (e; E14.5) or 3 days (f; E16.5). Femur sections were stained for nuclei and CD31. Asterisks: Tomato+ chondrocytes and sharps: Tomato+ cells in primary ossification center. Red: tdTomato, blue: Alexa633, gray: DAPI.g–j. Lineage-tracing of embryonic Col2-creER+ and Osx-creER+ cells was performed by injecting 1mg tamoxifen into pregnant mice at E13.5, and Col2-creER; R26RTomato (g,h) or Osx-creER; R26RTomato (i,j) mice were chased until indicated postnatal day (P0 or P21). Shown are femur sections with the distal side on the left. Col2creER-E13.5 represents descendants of Col2-creER+ cells at E13.5. White indicates tdTomato. Scale bars: 100µm (a,b,e,f) or 500µm (c,d,g–j).

Fig 4: Macrophage GR as a crucial regulator of myofibroblast differentiation in the infarct microenvironment. A, B) FACS strategy to obtain highly purified fibroblasts and myofibroblasts from GRflox (A) and GRLysMCre (B) ischemic myocardium 3 d after coronary artery ligation. Endothelial, hematopoietic, and vascular cells were excluded by selecting cells that are CD31-/TER-119-, CD45-/CD11b-, and NG2-/PDGFRß-. Immunocytochemical staining of sorted MEFSK4+ cells showing vimentin (Vim) and a-SMA. Scale bars = 50 µm. C) RT-PCR was used to detect the relative gene expression of a-SMA (Acta2), MMP-2, periostin (Postn), collagen I a1 (Col1a1), platelet-derived growth factor receptor a (Pdgfra), and discoidin domain-containing receptor 2 (Ddr2). D) Zymography and reverse zymography of conditioned medium from cardiac fibroblasts isolated from GRflox/GRLysMCre infarcts. Means ± sem (n = 3–4). *P < 0.05 vs. GRflox.

Fig 5: Platelet depletion does not affect neuronal density, microthrombi formation or atrophy in the chronic phase after ischaemic stroke. Disregarding differences between contra- and ipsilesional hemispheres, both treatment groups showed the same levels of CD31+/GPIX+ vessel staining (A, B) as well as NeuN+ (C) neuronal staining. Atrophy of brain tissue was calculated based on scans from brain slices at the end of the experiment on day 28 (D). No difference occurred between platelet depleted and control antibody treated animals. Representative images were taken from each group to show infarct size and atrophy (E). Infarcts are visible as brighter areas in MRI scans, located in the striatum. Unstained brain scans from day 28 post ischaemia show bright areas in rostral slices as a residue from the infarcted area. In case of normal distribution unpaired, two-tailed Student's t-test corrected by Holm-Sidak method was used for statistical analysis, otherwise Mann-Whitney-U test was applied. Scale bar indicates 50 µm [B]; 1 cm [E]. N = 5/7 (Atrophy), N = 5/6 (Immunohistochemistry).