Fig 2: CCR2-KO BMDMs reconstitution reduces infiltration into the brain and reverses the effects of HDAC3-miKO. (A) Experimental design indicating the strategy for CCR2 deficency macrophages administration. (B) Flow cytometry showing depletion of macrophages 24 h after CLO administration. (C-D) Representative images of F4/80/P2RY12 immunostaining and (D) quantification of the number of F4/80+P2RY12‑ cells. (E-G) Sensorimotor function assessment by Garcia score (E) and foot fault test (F&G). n = 6 for WT-tFCI+WT-BMDM or miKO-tFCI+WT-BMDM; n = 11 for miKO-tFCI+CCR2 KO-BMDM. (H) Representative images of dMBP immunofluorescence staining and the corresponding 3D rendering in three groups. (I) Quantification of dMBP volume (μm3) per mm3. (J) Quantification of dMBP area fraction (%). All data are presented as means±SEM. Data were analyzed using (D&I-J) one-way ANOVA followed by Bonferroni's post hoc test or (E-G) two-way ANOVA followed by Bonferroni's post hoc test. Comparisons in the yellow blocks referred to the main effects between groups for the ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001 for comparison between miKO-tFCI+WT-BMDM and miKO-tFCI + CCR2-KO BMDM; #p < 0.05; ##p < 0.01 for comparison between WT-tFCI + WT-BMDM and miKO-tFCI + WT-BMDM.

Fig 3: HDAC3-deficient microglia recruit macrophage reinforcements to the infarct core. (A) Macrophages (CD11b+CD45hiLy6C+), neutrophils (CD11b+CD45hiCD11c-Gr1+), dendritic cells (DCs) (CD11b+CD45hiCD11c+Gr1-), T cells (CD11b-CD45hiCD3+), B cells (CD11b-CD45hiCD19+) and microglia (CD11b+CD45int). (B-H) The percentage of these cells to single cells, respectively. n = 8/group. (I) Representative large image of Iba1/NeuN immunostaining, which indicated the infarct core and penumbra region photographed for Iba1/F4/80/P2RY12 staining (J-M). (J&K) Representative images of Iba1/P2RY12 immunostaining (J) and quantification of the number of Iba1+P2RY12- cells and Iba1+P2RY12+ cells (K) in the ischemic penumbra 3 d after tFCI. The right panel showed the proportion of Iba1+P2RY12- cells or Iba1+P2RY12+ cells to the overall Iba1+ cells. (L&M) Representative images of F4/80/P2RY12 immunostaining (L) and quantification of the number of F4/80+P2RY12- cells and P2RY12+ cells (M) in the ischemic core 3 d after tFCI. The right panel showed the proportion of F4/80+P2RY12- cells or P2RY12+ cells to the overall F480+ or P2RY12+ cells. n = 4 for WT-tFCI; n = 5 for miKO-tFCI. All data are presented as means±SEM. Data were analyzed using (B-E) one-way ANOVA followed by Bonferroni's post hoc, (F-H) Kruskal-Wallis test followed by Dunn's post hoc test or (K&M) unpaired two-tailed Student's t test. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance, as indicated (B-H). ***p < 0.001 for comparison of Iba1+P2RY12+ cell number between miKO-tFCI and WT-tFCI. ##p < 0.01 for comparison of the overall Iba1+ cells (K). ***p < 0.001 for comparison of F4/80+P2RY12- cell number between miKO-tFCI and WT-tFCI. ##p < 0.01 for comparison of the overall F480+ or P2RY12+ cells (M).

Fig 4: Microglia closely interact with endothelial cells and Müller cells during inner retinal vascular development. (A) Representative P10 retinal cryosection immunostained for glutamine synthetase (GS), IsolectinB4 (IB4), and Iba1 revealing microglial association with the growing inner retinal blood vessels and Müller cells, (arrows). (B) Representative P10 retinal flatmount immunostained for P2ry12 and CD31 showing microglial association with the growing intermediate and deep vascular plexuses (n = 4). Bar graph quantifies the number of P2ry12+ cells in the deep and intermediate vascular plexuses at P10 (n = 4). (C) Representative P10 retinal flatmounts from control and PLX5622-treated groups showing microglial (P2ry12) distribution. Bar graph quantifies the number of P2ry12+ cells in the control and microglia depleted (PLX5622) groups and (n = 3). (D) Magnified representative P10 retinal flatmounts from control and PLX5622-treated groups showing microglial (P2ry12) immunostaining (n = 3). Scale bars: (A) is 75 μm, (B) is 50 μm, and (C) is 500 μm