Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome methods
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Materials and Methods
All experiments were performed in fms-EGFP (MacGreen) mice, except where indicated, in which all microglia express the fluorescent reporter (; ). KO mice were provided by Beata Sperlagh, Institute of Experimental Medicine (P2Y12 KO) and Greg Lemke, Salk Institute (MerTK/Axl KO). Microglial-specific, inducible MerTK/Axl mice were generated using Cx3cr1 CreER ( ) and Mertk fl/fl ( ), described previously. To induce deletion of the Mertk fl/fl allele in Cx3cr1 CreER /+ Mertk fl/fl mice, two doses of tamoxifen dissolved in corn oil (75 mg/kg) or corresponding volume of corn oil alone (control mice) were administered intraperitoneally at postnatal days (P)21 and P23. All mice used were in a C57BL/6 background. Mice were housed in 12 h light/dark cycle with ad libitum access to food and water. Mice received a single dose of 5-bromo-2′-deoxyuridine (BrdU; 100-150 mg/kg) at P28....
Immunofluorescence.
Six series of 50-µm-thick coronal sections of mouse brains were cut using a Leica VT 1200S vibrating blade microtome (Leica Microsystems). Fluorescent immunostaining was performed following standard procedures (; ). Free-floating vibratome sections were blocked in permeabilization solution (0.3% Triton X-100, 0.5% BSA in PBS; all from Sigma-Aldrich) for 3 h at room temperature (RT), and then incubated overnight with the primary antibodies diluted in the permeabilization solution at 4°C. For BrdU labeling an antigen retrieval procedure was performed by incubating in 2 m HCl for 30 min at 37°C and then washing with 0.1 m sodium tetraborate for 10 min at RT before the blockade of the sections. After overnight incubation with primary antibodies, brain sections were thoroughly washed with 0.3% Triton in PBS. Next, the sections were incubated with fluorochrome-conjugated sec...
Immunofluorescence.
Primary microglial cultures were fixed for 10 min in 4% PFA and then transferred to PBS. Fluorescent immunostaining was performed following standard procedures (; ). Coverslips with primary microglial cultures were blocked in 0.1% Triton X-100, 0.5% BSA in PBS for 30 min at RT. The cells were then incubated with primary antibodies in permeabilization solution (0.2% Triton X-100, 0.5% BSA in PBS) for 1 h at RT, rinsed in PBS and incubated in the secondary antibodies containing DAPI (5 mg/ml) in the permeabilization solution for 1 h at RT. After washing with PBS, primary cultures were mounted on glass slides with Dako Cytomation Fluorescent Mounting Medium.
Western blot.
CM-treated NPCs were directly lysed in RIPA buffer containing protease and phosphatase inhibitor cocktail (100 ×; ThermoFisher Scientific). Cells were sonicated for 5 s and then centrifuged (10,000 × g, 10 min). Solubilized protein was quantified in triplicates by BCA Assay Kit (ThermoFisher Scientific) at 590 nm using a microplate reader (Synergy HT, BioTek). Ten to 15 µg of protein (denatured with β-mercaptoethanol) were loaded onto Tris-glycine gradient polyacrylamide gels (8-16%; ThermoFisher Scientific) and run at 120 V for 90 min. Protein samples were then blotted to nitrocellulose membranes (0.45 µm pore size; ThermoFisher Scientific) at 220 mA for 2 h. Transfer efficiency was verified by Ponceau S (Sigma-Aldrich) staining. For immunoblotting, membranes were rinsed in Tris-Buffered Saline containing 0.1% Tween 20 (TBS-T; Sigma-Aldrich) and then bl...
Statistical analysis of gene expression arrays.
To analyze the differential expression between naive ( t = 0) and phagocytic ( t = 3 h and t = 24 h) microglia groups over time the statistical analysis the maSigPro package of R/Bioconductor was used (R v3.0.3, Bioconductor release v2.13, maSigPro v1.34.1; ). This method is based on a general regression approximation for the modeling and adjustment of the parameters required according to the type of analysis. The parameter "Time" is considered as a continuous variable, and creates a regression model of the gene response. The analysis was performed in three steps. First, genes that exhibited changes in expression over time were selected based on a p -corrected Benjamini-Hochberg (FDR) value. Next, for each of the genes that presented a significant change in their expression over time a regression was applied to determine their model ( R 2 > 0.7) to identify patterns...
Measurement outputs
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Protein expression by western blot
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
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- Band quantification and normalized densitometry values
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- Relative expression values or fold-change comparisons across groups
Microscopy-based tissue or cell-state imaging
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Quantify the primary readouts for this experiment: Protein expression by western blot; Microscopy-based tissue or cell-state imaging.
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Report representative outputs alongside summary comparisons for Protein expression by western blot, Microscopy-based tissue or cell-state imaging.
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Evidence quotes (5)
All experiments were performed in fms-EGFP (MacGreen) mice, except where indicated, in which all microglia express the fluorescent reporter (; ). KO mice were provided by Beata Sperlagh, Institute of Experimental Medicine (P2Y12 KO) and Greg Lemke, Salk Institute (MerTK/Axl KO). Microglial-specific, inducible MerTK/Axl mice were generated using Cx3cr1 CreER ( ) and Mertk fl/fl ( ), described previously. To induce deletion of the Mertk fl/fl allele in Cx3cr1 CreER /+ Mertk fl/fl mice, two doses of tamoxifen dissolved in corn oil (75 mg/kg) or corresponding volume of corn oil alone (control mice) were administered intraperitoneally at postnatal days (P)21 and P23. All mice used were in a C57BL/6 background. Mice were housed in 12 h light/dark cycle with ad libitum access to food and water. Mice received a single dose of 5-bromo-2′-deoxyuridine (BrdU; 100-150 mg/kg) at P28. At 24 h or 28 d after BrdU injection, mice were anesthetized with a mixture of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively), perfused with 20 U/ml heparin in PBS followed by 4% PFA in PBS. Brains were collected, immersion fixed for 4 h in 4% PFA in PBS, and stored in 30% sucrose, 30%...
Six series of 50-µm-thick coronal sections of mouse brains were cut using a Leica VT 1200S vibrating blade microtome (Leica Microsystems). Fluorescent immunostaining was performed following standard procedures (; ). Free-floating vibratome sections were blocked in permeabilization solution (0.3% Triton X-100, 0.5% BSA in PBS; all from Sigma-Aldrich) for 3 h at room temperature (RT), and then incubated overnight with the primary antibodies diluted in the permeabilization solution at 4°C. For BrdU labeling an antigen retrieval procedure was performed by incubating in 2 m HCl for 30 min at 37°C and then washing with 0.1 m sodium tetraborate for 10 min at RT before the blockade of the sections. After overnight incubation with primary antibodies, brain sections were thoroughly washed with 0.3% Triton in PBS. Next, the sections were incubated with fluorochrome-conjugated secondary antibodies and DAPI (5 mg/ml; Sigma-Aldrich) diluted in the permeabilization solution for 3 h at RT. After washing with PBS, the sections were mounted on glass slides with Dako Cytomation Fluorescent Mounting Medium.
Primary microglial cultures were fixed for 10 min in 4% PFA and then transferred to PBS. Fluorescent immunostaining was performed following standard procedures (; ). Coverslips with primary microglial cultures were blocked in 0.1% Triton X-100, 0.5% BSA in PBS for 30 min at RT. The cells were then incubated with primary antibodies in permeabilization solution (0.2% Triton X-100, 0.5% BSA in PBS) for 1 h at RT, rinsed in PBS and incubated in the secondary antibodies containing DAPI (5 mg/ml) in the permeabilization solution for 1 h at RT. After washing with PBS, primary cultures were mounted on glass slides with Dako Cytomation Fluorescent Mounting Medium.
CM-treated NPCs were directly lysed in RIPA buffer containing protease and phosphatase inhibitor cocktail (100 ×; ThermoFisher Scientific). Cells were sonicated for 5 s and then centrifuged (10,000 × g, 10 min). Solubilized protein was quantified in triplicates by BCA Assay Kit (ThermoFisher Scientific) at 590 nm using a microplate reader (Synergy HT, BioTek). Ten to 15 µg of protein (denatured with β-mercaptoethanol) were loaded onto Tris-glycine gradient polyacrylamide gels (8-16%; ThermoFisher Scientific) and run at 120 V for 90 min. Protein samples were then blotted to nitrocellulose membranes (0.45 µm pore size; ThermoFisher Scientific) at 220 mA for 2 h. Transfer efficiency was verified by Ponceau S (Sigma-Aldrich) staining. For immunoblotting, membranes were rinsed in Tris-Buffered Saline containing 0.1% Tween 20 (TBS-T; Sigma-Aldrich) and then blocked for 1 h in TBS-T containing 5% powder milk. Membranes were afterward incubated with rabbit primary antibodies for REST (1:500; EMD, Millipore), and phosphorylated Smad 1/5/9 (1:500; Cell Signaling Technology), and mouse primary antibodies for Smad 1 (1:500; Santa Cruz Biotechnology), Ascl1 (1:...
To analyze the differential expression between naive ( t = 0) and phagocytic ( t = 3 h and t = 24 h) microglia groups over time the statistical analysis the maSigPro package of R/Bioconductor was used (R v3.0.3, Bioconductor release v2.13, maSigPro v1.34.1; ). This method is based on a general regression approximation for the modeling and adjustment of the parameters required according to the type of analysis. The parameter "Time" is considered as a continuous variable, and creates a regression model of the gene response. The analysis was performed in three steps. First, genes that exhibited changes in expression over time were selected based on a p -corrected Benjamini-Hochberg (FDR) value. Next, for each of the genes that presented a significant change in their expression over time a regression was applied to determine their model ( R 2 > 0.7) to identify patterns or models of change based on time variables, obtaining 20,800 probes. Finally, the probes were selected according to their fit to the regression model. Next, the number of genes with a very high differential pattern was reduced by applying a more restrictive criterion ( R 2 > 0.9), obtaining 13,146....
Machine-readable layer
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