Journal: The EMBO Journal

Article Title: Microglial colonization of the developing mouse brain is controlled by both microglial and neural CSF-1

doi: 10.1038/s44318-025-00625-8

Figure Lengend Snippet: ( A ) Immunolabeling of coronal brain sections from E14.5 Cx3cr1 gfp/+ embryos showing co-expression of GFP, MAC2 and OPN at the CSA ( n = 3). Close-ups are 75 µm wide. ( B ) Immunolabeling of coronal brain sections from E14.5 Cx3cr1 gfp/+ embryos showing co-expression of GFP and GPNMB at the CSA ( n = 4). ( C ) Immunolabeling of coronal brain sections from P7 Cx3cr1 gfp/+ pups showing co-expression of GFP, MAC2 and OPN at the EDWM ( n = 3). ( D ) Immunolabeling of coronal brain sections from P7 Cx3cr1 gfp/+ pups showing co-expression of GFP and GPNMB at the EDWM ( n = 3). ( E ) Immunolabeling of coronal brain sections from E14.5 Cx3cr1 gfp/+ embryos showing co-expression of GFP and EdU (open arrowheads) at the CSA. ( F ) Percentage of OPN-positive ameboid microglia at the CSA from E14.5 embryos ( n = 13; from at least two distinct litters). ( G ) Percentage of EdU-positive microglia at the CSA at E14.5 amongst OPN-positive or OPN-negative ameboid microglia ( n = 9; from one litter) ( P = 0.0197). ( H ) Immunolabeling of coronal brain sections from P7 Cx3cr1 gfp/+ embryos showing co-expression of GFP and EdU (open arrowheads) at the EDWM. ( I ) Percentage of OPN-positive ameboid microglia at the EDWM from P7 pups ( n = 13; from at least two distinct litters). ( J ) Percentage of EdU-positive ameboid microglia at the EDWM at P7 amongst OPN-positive or OPN-negative ameboid microglia ( n = 8; from two distinct litters) ( P = 0.0272). Data were presented as mean ± SEM. Two-sided unpaired Mann–Whitney test were performed to assess differences ( G , J ). * P < 0.05. Scale bars: 100 µm ( A – D , H ); 50 µm ( E ). Close-ups are 100 µm ( A – D ) and 75 µm ( E , H ) wide. CSA cortico-striato-amygdalar boundary, EDWM early-dorsal white matter, H Hoechst. See Figs. and . .

Article Snippet: Slices were first incubated for 1 h at room temperature (RT) in 0.2% Triton X-100, 0.2% Gelatin in PBS (blocking solution), and then incubated in the same blocking solution with the following primary antibodies overnight at 4 °C: rat anti-CD206 (1/200; Bio-Rad Cat# MCA2235, RRID:AB_324622), chicken anti-GFP (1/1000; Aves Labs Cat# GFP-1020, RRID:AB_10000240), rabbit anti-IBA1 (1/500; FUJIFILM Wako Shibayagi Cat# 019-19741, RRID:AB_839504), chicken anti-IBA1 (1/400; Synaptic Systems Cat# 234 009, RRID:AB_2891282), rabbit anti-KI67 (1/200; Abcam Cat# ab15580, RRID:AB_443209), rat anti-Lyve1 (ALY7) (1/200; Thermo Fisher Scientific Cat# 14-0443-82, RRID:AB_1633414), rat anti-GAL3 (MAC2) (1/1000; Cedarlane Cat# CL8942AP; RRID:AB_10060357), goat anti-OPN (1/400; R and D Systems Cat# AF808, RRID:AB_2194992), goat anti-mouse Osteoactivin (GPNMB) (1/200; R and D Systems Cat# AF2330; RRID:AB_2112934), rabbit anti-P2RY12 (1/500; AnaSpec; EGT Group Cat# 55043 A, RRID:AB_2298886), and anti-VGlut2 (1/2000; Millipore Cat# AB2251-I, RRID:AB_2665454).

Techniques: Immunolabeling, Expressing, MANN-WHITNEY

Journal: The EMBO Journal

Article Title: Microglial colonization of the developing mouse brain is controlled by both microglial and neural CSF-1

doi: 10.1038/s44318-025-00625-8

Figure Lengend Snippet: ( A ) Immunolabeling of coronal brain sections showing comparable co-expression of GFP, MAC2, and OPN at the CSA in E14.5 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos (tamoxifen administered at E12.5) ( n control = 5; n mutant = 4). ( B ) Immunolabeling of coronal brain sections showing comparable co-expression of GFP and GPNMB at the CSA in E14.5 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos (tamoxifen administered at E12.5). ( C ) Immunolabeling of coronal brain sections showing comparable co-expression of GFP, MAC2 and OPN at the CSA in P7 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos (tamoxifen administered at P3) ( n control = 3; n mutant = 3). ( D ) Immunolabeling of coronal brain sections showing comparable co-expression of GFP and GPNMB at the CSA in P7 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos (tamoxifen administered at P3). ( E ) Immunolabeling of coronal brain sections from control and Cx3cr1 creER/+ ;Csf1 fl/fl mice showing absence of expression of OPN in cortical microglia (IBA1-positive) at P7 (tamoxifen administered at P3) while some white matter microglia do express OPN (solid arrowheads). Close-ups are 200 µm wide. ( F ) Proportion of proliferative microglia in control and Cx3cr1 creER/+ ;Csf1 fl/fl neocortex of P7 mice (tamoxifen administered at P3)( n control = 3 (2 F;1 M) ; n mutant = 4 (2 F;2 M); from one litter). ( G ) Microglial density (IBA1-positive cells/mm²) in control and Cx3cr1 creER/+ ;Csf1 fl/fl neocortex of P7 mice (tamoxifen administered at P3)( n control = 3 (2 F;1 M) ; n mutant = 4 (2 F;2 M); from one litter). ( H ) RNAscope experiments on coronal brain sections from P7 control and Cx3cr1 creER/+ ;Csf1 fl/fl pups showing Csf1 expression in IBA1-positive ameboid microglia at the CSA (solid arrowheads) after tamoxifen administration at P1 and P3. ( I ) Normalized quantification of RNAscope experiments on coronal brain sections from P7 control and Cx3cr1 creER/+ ;Csf1 fl/fl embryos showing decreased Csf1 expression in mutant mice. Left column shows all quantified cells and right column the mean expression per animal ( n control = 3 (1 F,2 M); n mutant = 3 (2 M,1 F); from two distinct litters). ( J ) Immunolabeling of coronal brain sections from control and Cx3cr1 creER/+ ;Csf1 fl/fl mice showing co-expression of IBA1 and OPN in ameboid microglia within the EDWM accumulation at P7 (tamoxifen administered at P1 and P3) ( n control = 3 (1 F,1 M) ; n mutant = 3 (2 F;1 M); from one litter)(all cells, left: P < 0.0001; mean per animal, right: P = 0.10). ( K ) Microglial density (IBA1-positive cells/mm²) in control and Cx3cr1 creER/+ ;Csf1 fl/fl EDWM of P7 mice (tamoxifen administered at P1 and P3) ( n control = 9 (3 F;6 M); n mutant = 6 (3 F;3 M); from three distinct litters) ( P = 0.0028). ( L ) Number of OPN-positive microglia at the EDWM at P7 ( n control = 9 (3 F;6 M); n mutant = 6 (3 F;3 M); from three distinct litters) ( P = 0.0016). Data were presented as mean ± SEM. Two-sided unpaired Mann–Whitney test were performed to assess differences ( F , G , J – L ). ns not significant, ** P < 0.01, *** P < 0.001. Scale bars: 100 µm ( A – D , H , I ); 200 µm ( E ). CSA cortico-striatal-amygdalar boundary, EDWM early-dorsal white matter, H Hoechst, Ncx neocortex.

Article Snippet: Slices were first incubated for 1 h at room temperature (RT) in 0.2% Triton X-100, 0.2% Gelatin in PBS (blocking solution), and then incubated in the same blocking solution with the following primary antibodies overnight at 4 °C: rat anti-CD206 (1/200; Bio-Rad Cat# MCA2235, RRID:AB_324622), chicken anti-GFP (1/1000; Aves Labs Cat# GFP-1020, RRID:AB_10000240), rabbit anti-IBA1 (1/500; FUJIFILM Wako Shibayagi Cat# 019-19741, RRID:AB_839504), chicken anti-IBA1 (1/400; Synaptic Systems Cat# 234 009, RRID:AB_2891282), rabbit anti-KI67 (1/200; Abcam Cat# ab15580, RRID:AB_443209), rat anti-Lyve1 (ALY7) (1/200; Thermo Fisher Scientific Cat# 14-0443-82, RRID:AB_1633414), rat anti-GAL3 (MAC2) (1/1000; Cedarlane Cat# CL8942AP; RRID:AB_10060357), goat anti-OPN (1/400; R and D Systems Cat# AF808, RRID:AB_2194992), goat anti-mouse Osteoactivin (GPNMB) (1/200; R and D Systems Cat# AF2330; RRID:AB_2112934), rabbit anti-P2RY12 (1/500; AnaSpec; EGT Group Cat# 55043 A, RRID:AB_2298886), and anti-VGlut2 (1/2000; Millipore Cat# AB2251-I, RRID:AB_2665454).

Techniques: Immunolabeling, Expressing, Control, Mutagenesis, RNAscope, MANN-WHITNEY