Figure 2.
Widespread engraftment and persistence of transplanted G795A but not WT CIMs in the PLX3397-treated brain. (A) Experimental paradigm depicting the timing of neonatal CIM injection, PLX treatment (290 mg/kg chow) and endpoints at P14 (“On PLX”) and P21 (“Off PLX”). (B) Representative rendering of donor cell distribution and mean engraftment ± SEM, 14 d after cell injection, with continuous PLX treatment. Box i denotes field shown in F. Scale bar, 1,000 µm. (C) Representative rendering showing donor cell distribution 21 d after cell injection. PLX was stopped 7 d prior to harvest. Boxes ii and iii denote fields shown in F. (D) Quantification of brain engraftment by G795A-CIMs in hosts on or off PLX, and WT-CIMs in hosts on PLX. G795A-On PLX: n = 3; G795A-Off Plx: n = 3; WT-On Plx: n = 4. One-way ANOVA (P = 0.0001). (E) Quantification of donor cell density in cortex. G795A-On PLX: n = 3; G795A-Off Plx: n = 3; WT-On Plx: n = 4. One-way ANOVA (P = 0.0070). Dotted gray line represents average endogenous IBA1+ cells from n = 3 age-matched controls. (F) Representative images of G795A- and WT-CIMs following neonatal transplant, showing donor cell GFP (green), IBA1 immunostaining (red), and DAPI (blue) On and Off PLX. Row 4 displays endogenous microglia repopulation after PLX removal from box iii in C. Scale bar, 50 µm. (G) Experimental paradigm depicting timing of adult CIM injection, PLX treatment, and endpoints at D14 (On PLX) and D21 (Off PLX). (H) Representative rendering of G795A-CIM distribution and mean engraftment ± SEM, 14 d after cell injection, with continuous PLX treatment. Scale bar, 1,000 µm. (I) Representative image of G795A-CIM distribution and mean engraftment ± SEM, 21 d after cell injection. PLX was stopped 7 d prior to harvest. Scale bar, 1,000 µm. (J) Quantification of cortical engraftment area by G795A and WT donor CIMs. G795A-On PLX: n = 6; G795A-Off Plx: n = 3; WT-On Plx: n = 5. One-way ANOVA (P < 0.0001). (K) Quantification of donor cell density in cortex. One-way ANOVA (P = 0.0019). (L) Representative images of G795A and WT CIMs following adult transplant, showing donor cell GFP (green), IBA1 immunostaining (red), and DAPI (blue) both On (rows 1 and 2) and Off PLX (row 3). Scale bar, 50 µm. (M) Top: Representative confocal image of G795A-CIM morphology (green), IBA1 immunostaining (red), TMEM119 immunostaining (white), and DAPI (blue). Scale bar, 25 µm. Bottom: Representative confocal image of G795A-CIM morphology (green), IBA1 immunostaining (red), Ms4a7 in situ (white), and DAPI (blue). Scale bar, 25 µm. In D, E, J, and K, dots represent biological replicates, each the average of three matched sagittal sections. Error bars represent SEM. P values calculated as Tukey’s HSD: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Widespread engraftment and persistence of transplanted G795A but not WT CIMs in the PLX3397 - treated brain. (A) Experimental paradigm depicting the timing of neonatal CIM injection, PLX treatment (290 mg/kg chow) and endpoints at P14 (“On PLX”) and P21 (“Off PLX”). (B) Representative rendering of donor cell distribution and mean engraftment ± SEM, 14 d after cell injection, with continuous PLX treatment. Box i denotes field shown in F. Scale bar, 1,000 µm. (C) Representative rendering showing donor cell distribution 21 d after cell injection. PLX was stopped 7 d prior to harvest. Boxes ii and iii denote fields shown in F. (D) Quantification of brain engraftment by G795A-CIMs in hosts on or off PLX, and WT-CIMs in hosts on PLX. G795A-On PLX: n = 3; G795A-Off Plx: n = 3; WT-On Plx: n = 4. One-way ANOVA (P = 0.0001). (E) Quantification of donor cell density in cortex. G795A-On PLX: n = 3; G795A-Off Plx: n = 3; WT-On Plx: n = 4. One-way ANOVA (P = 0.0070). Dotted gray line represents average endogenous IBA1+ cells from n = 3 age-matched controls. (F) Representative images of G795A- and WT-CIMs following neonatal transplant, showing donor cell GFP (green), IBA1 immunostaining (red), and DAPI (blue) On and Off PLX. Row 4 displays endogenous microglia repopulation after PLX removal from box iii in C. Scale bar, 50 µm. (G) Experimental paradigm depicting timing of adult CIM injection, PLX treatment, and endpoints at D14 (On PLX) and D21 (Off PLX). (H) Representative rendering of G795A-CIM distribution and mean engraftment ± SEM, 14 d after cell injection, with continuous PLX treatment. Scale bar, 1,000 µm. (I) Representative image of G795A-CIM distribution and mean engraftment ± SEM, 21 d after cell injection. PLX was stopped 7 d prior to harvest. Scale bar, 1,000 µm. (J) Quantification of cortical engraftment area by G795A and WT donor CIMs. G795A-On PLX: n = 6; G795A-Off Plx: n = 3; WT-On Plx: n = 5. One-way ANOVA (P < 0.0001). (K) Quantification of donor cell density in cortex. One-way ANOVA (P = 0.0019). (L) Representative images of G795A and WT CIMs following adult transplant, showing donor cell GFP (green), IBA1 immunostaining (red), and DAPI (blue) both On (rows 1 and 2) and Off PLX (row 3). Scale bar, 50 µm. (M) Top: Representative confocal image of G795A-CIM morphology (green), IBA1 immunostaining (red), TMEM119 immunostaining (white), and DAPI (blue). Scale bar, 25 µm. Bottom: Representative confocal image of G795A-CIM morphology (green), IBA1 immunostaining (red), Ms4a7 in situ (white), and DAPI (blue). Scale bar, 25 µm. In D, E, J, and K, dots represent biological replicates, each the average of three matched sagittal sections. Error bars represent SEM. P values calculated as Tukey’s HSD: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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