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. 2020 Jun 23;9:e54590. doi: 10.7554/eLife.54590

Figure 1. Microglia recombination by P2ry12-CreER.

(A) Design of the P2ry12 knock-in allele and a P2ry12-CreER;Rosa26Ai14 mouse brain section immunostained for P2RY12 (cyan) and TdTomato (red). (B) Flow cytometry analysis of recombined TdTomato+ microglia in a P2ry12-CreER;Rosa26Ai14 mouse. Microglia were pre-gated on forward scatter, isolation of single cells, and removal of dead cells. TdTomato+ cells are marked in red. 94.9 ± 2.75% of microglia (CD11b+CD45int cells) were recombined by P2ry12-CreER. (C-E) Images of recombination in microglia of the cerebral cortex (C), spinal cord (D) cerebellum (E) in P2ry12-CreER; Rosa26Ai14 mice. Sections stained with pan-macrophage marker IBA1 (green). (F) Immunohistochemical quantification of recombination in the cerebral cortex, spinal cord, hippocampus and cerebellum. For B-E, n = 3 mice. Error bars in F = standard error of the mean (SEM). Scale bars = 20 µm (A), 50 µm (C).

Figure 1—source data 1. Microglial recombination by P2ry12-CreER.

Figure 1.

Figure 1—figure supplement 1. qPCR and western blot analysis of P2ry12 expression; background recombination of P2ry12-CreER.

Figure 1—figure supplement 1.

(A) Quantitative RT-PCR analysis of P2ry12 brain transcript levels in wildtype, P2ry12-CreER heterozygous and P2ry12-CreER homozygote mouse brains. (B) Western blot analysis of P2RY12 protein expression in WT and P2ry12-CreER homozygote brains, with or without tamoxifen treatment. (C) Densitometry quantification of western blot analysis. (D) Non-tamoxifen recombination in the cerebral cortex of a P2ry12-CreER; Rosa26Ai14 heterozygous mouse. Two recombined microglia are seen in this field of view of the cerebral cortex. Section stained for IBA1 (green). (E) High-magnification images of IBA1 staining in wildype (E), P2ry12-CreER heterozygous (E’) and P2ry12-CreER homozygous (E’) mice. For analysis in A and C, N = 3 mice per condition/genotype. For analysis in A., **p-value=0.0089. Statistical comparisons in A. and C. were made with a one-way ANOVA and a Tukey’s multiple comparisons test. Error bars = SEM (A), SE (C). Scale bars = 500 µm (D), 20 µm (E).
Figure 1—figure supplement 2. Flow cytometry gating strategy used for isolating microglia and TdT+ cells.

Figure 1—figure supplement 2.

Flow cytometry analysis of microglial recombination in P2ry12-CreER; Rosa26Ai14 brains involved pre-gating for 1) Side-scatter 2) Single cells 3) Live cells. Microglia were then identified by expression of CD11b and CD45 (4) and analyzed for TdT expression (5).