Journal: Nature Communications

Article Title: Microglial debris is cleared by astrocytes via C4b-facilitated phagocytosis and degraded via RUBICON-dependent noncanonical autophagy in mice

doi: 10.1038/s41467-022-33932-3

Figure Lengend Snippet: a Scheme of in vivo debris engulfment examinations by microglial depletion. b Microglial debris is unlikely to be phagocytosed by pericytes, endothelial cells, VSMCs, OPCs, oligodendrocytes, neurons or NSCs in vivo. NESTIN-GFP is illustrated by the red pseudocolor for better visualization. Astrocytes (GFAP): 0.24 ± 0.05%, pericytes (PDGFR-β): 0.03 ± 0.01%, endothelial cells (CD31): 0.03 ± 0.01%, VSMCs (α-SMA): 0.03 ± 0.02%, OPCs (PDGFR-α): 0.08 ± 0.02%, oligodendrocytes (MBP): 0.05 ± 0.01%, oligodendrocytes (CC1): 0.07 ± 0.02%, neurons (TUJ1): 0.04 ± 0.01%, NSCs (NESTIN-GFP): 0.03 ± 0.01%. N = 4 mice in the NESTIN-GFP group and N = 5 mice in the rest of the groups. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). P = 3.1086e–015, 4.6629e–015, 2.6645e–015, 1.0612e–012, 3.4639e–014, 9.7167e–013, 4.9960e–015, and 1.2434e–014. c Scheme of the in vivo BBB integrity examination with 10-kDa dextran-FITC. d Confocal images show that the BBB is not compromised during microglial depletion in vivo. Each experiment was independently repeated from 4 mice with similar results. PLX5622: PLX5622-formulated AIN-76A diet; VSMCs vascular smooth muscle cells, OPC oligodendrocyte precursor cell, OL oligodendrocyte, NSC neural stem cell, IV intravenous. The source data are provided as a Source Data file.

Article Snippet: The primary antibodies used in this study included the following: goat anti-mCherry (Biorbyt, Cat#: Orb11618, Lot#: J2446; 1:500), rat anti-CD31 (BD Biosciences, Cat#: 550274, Lot#: 9259767; 1:10), rabbit anti-IBA1 (Wako, Cat#: 019-19741 Lot#: WDK2121; 1:500), goat anti-IBA1 (Abcam, Cat#: ab5076, Lot#: GR3187278-2; 1:500), chicken anti-GFP (Abcam, Cat#: ab13970, Lot#: GR236651-12; 1:1,000), rabbit anti-S100β (Abcam, Cat#: ab52642, Lot#: GR252937-5; 1:300), rabbit anti-GFAP (Abcam, Cat#: ab7260, Lot#: GR297722-2; 1:300), rabbit anti-α-SMA (Abcam, Cat#: ab124964, Lot#: GR181740-66; 1:300), mouse anti-GFAP (Sigma‒Aldrich, Cat#: G3893, Lot#: 105M4784 V; 1:200), goat anti-PDGFR-β (R&D systems, Cat#: AF385, Lot#: BIWO619121; 1:200), rabbit anti-LC3 (Cell Signaling Technologies, Cat#: 4108, Lot#: 3; 1:100), rat anti-LAMP1 (1D4B) (Santa Cruz, Cat#: SC-19992, Lot#: C2715; 1:50), rabbit anti-PDGFR-α (Cell Signaling Technologies, Cat#: 3174 s, Lot#: 7; 1:500), mouse anti-CC1 (Millipore, Cat#: 14-0661-82, Lot#: 3129980; 1:200), rat anti-mouse C4 (Abcam, Cat#: ab11863, Lot#: GR3315169-2; 1:100), goat anti-GFP (Abcam, Cat#: AB6673, Lot#: GR3373716-2; 1:1,000), chicken anti-mCherry (Abcam, Cat#: ab205402, Lot#: GR3271744-8; 1:500), chicken anti-NESTIN (Abcam, Cat#: ab134017, Lot#: GR3291127-1; 1:200), rabbit anti-RFP (Abcam, Cat#: ab62341, Lot#: GR3319727-1; 1:1,000), rabbit anti-NeuN (Abcam, Cat#: ab177487, Lot#: GR3275122-6; 1:500), rabbit anti-MBP (Abcam, Cat#: ab40390, Lot#: GR297609-1; 1:200), rabbit anti-LAMININ (Sigma, Cat#: L9393-100UL, Lot#: 087M4889 V; 1:250), rabbit anti-GFP (Invitrogen, Cat#: A-11122, Lot#: 2273763; 1:1000), goat anti-OLIG2 (R&D, Cat#: AF2418, Lot#: UPA0719061; 1:400) and rabbit anti-PDGFα (Cell Signaling, Cat#: 3164 S, Lot#: 02/2020-6; 1:500).

Techniques: In Vivo