Table 1.
List of primary antibodies used for immunostaining.
Table 2.
List of secondary antibodies used for immunostaining.
Figure 1.
Distribution of TREM2 protein during postnatal development.
A). Schemes showing the regions quantified for TREM2 by immunohistochemistry: 1-Cortex; 2-Caudate-Putamen; 3-Rostral Corpus callosum; 4-subventricular zone; 5-Caudal corpus callosum; 6-fimbria; 7-hippocampal fissure and 8-thalamus. (B-F) Patterns of TREM2 expression were depicted as red dots in schemes showing rostral and caudal region of postnatal brains at P1 (B), P3 (C), P5 (D), P7 (E) and P10 (F).
Figure 2.
TREM2 immunostaining showing developmental modulation.
(A–D) Developmental changes in subventricular zone (svz) at P1 (A), P3 (B), P5 (C) and P7 (D), showing a reduction in TREM2 expression at P3 and then an increase from P5 to P7. (E–H) Developmental changes in caudal corpus callosum (cau cc) at P1 (E), P3 (F), P5 (G) and P7 (H), showing maximum TREM2 expression at P3 followed by a progressive reduction till P7. (I–L) Changes in fimbria (fim) at P1 (I), P3 (J), P5 (K) and P7 (L), with a maximum expression at P3. (M–P) Developmental expression of TREM2 in hippocampal fissure (hf) at P1 (M), P3 (N), P5 (O) and P7 (P), showing no modulation of TREM2. lv: lateral ventricle; hp: hippocampus; CA1:cornu ammonis 1. Scale bar = 50 µm.
Figure 3.
TREM2 expression was differentially modulated in a region and age dependent manner.
Quantification of TREM2+ area/mm2 in subventricular zone, SVZ (A); caudal corpus callosum, cc (B); rostral Corpus callosum (C); fimbria, FIM (D); hippocampal fissure, Hf (E); Cortex, Cx (F); Caudate Putamen, CP (G) and thalamus, Tl (H). Three to five animals were used at each time point. Data were represented as mean ± SEM. Analysis was performed using one-way ANOVA followed by Tukey post hoc test or Kruskal Wallis followed by multiple comparison tests as suitable. *p<0.05; **p<0.01; ***p<0.001.
Figure 4.
Expression of functional TREM2–DAP12 cells in postnatal brain.
Immunofluorescence study of TREM2 (green), coreceptor DAP12 (red), and nuclear stain DAPI (blue) was performed. Colocalization of TREM2 and DAP12 was found in subventricular zone, SVZ (A), corpus callosum, cc (B) and fimbria, fim (C). “Foamy” cells (arrowheads) and intracellular aggregation of TREM2 (arrows) was also observed. Colocalization can be seen in yellow. Scale bar A–C = 20 µm.
Figure 5.
TREM2 expression was found on microglia.
Triple immunofluorescence was performed to characterize the population of TREM2+ cells. (A–B) TREM2 colocalized with CD68+ cells, a marker of phagocytic activity but not with radial glia or astrocytes (GFAP+ cells) at P3 in subventricular zone, SVZ (A) and fimbria, fim (B). Insets beside the figures show separated channels: CD68 in red, TREM2 in green and GFAP in blue. (C–F) TREM2+ cells colocalized with Iba-1+ cells but not with blood vessels. At P1, all Iba-1+ cells expressed TREM2 in SVZ (C) and fimbria (E). The expression of TREM2 was restricted to a subpopulation of microglia at P7 (D and F). Insets in C–F represent separated channels: Iba1 in red, TREM2 in green and Tomato lectin (TL) in blue. Triple colocalization can be seen in purple. Scale bar A–B = 50 µm; C–F = 20 µm.
Figure 6.
Phenotypic characterization of TREM2+ microglia in subventricular zone.
TREM2 co-expression with: CD206 (A–B), CD16/32 (C–D), CD86 (E–F) and MHCII (G–H) was studied at P1 (A, C, E and G) and P7 (B, D, F and H). Insets beside each figure represent separated channels: CD206, CD16/32, CD86 and MHCII in red, TREM2 in green and Iba1 in blue. SVZ: subventricular zone. Triple colocalization can be seen in purple. Scale bar for A and B = 50 µm; scale bar for C–H = 20 µm.
Figure 7.
Phenotypic characterization of TREM2+ microglia in corpus callosum.
TREM2 co-expression with: CD206 (A–B), CD16/32 (C–D), CD86 (E–F) and MHCII (G–H) was studied at P1 (A, C, E and G) and P7 (B, D, F and H). Insets beside each figure represent separated channels: CD206, CD16/32, CD86 and MHCII in red, TREM2 in green and Iba1 in blue. cc: corpus callosum. Triple colocalization can be seen in purple. Scale bar for A and B = 50 µm; scale bar for C–H = 20 µm.
Figure 8.
Phenotypic characterization of TREM2+ microglia in hippocampal fissure.
TREM2 co-expression with: CD206 (A–B), CD16/32 (C–D), CD86 (E–F) and MHCII (G–H) was studied at P1 (A, C, E and G) and P7 (B, D, F and H). Insets beside each figure represent separated channels: CD206, CD16/32, CD86 and MHCII in red, TREM2 in green and Iba1 in blue. hf: hippocampal fissure. Triple colocalization can be seen in purple. Scale bar for A and B = 50 µm; C–H = 20 µm.
Figure 9.
Schematic summary of spatio–temporal changes in TREM2 phenotypes.
Developmental regulation of the TREM2+ phenotypes was shown in subventricular zone (A), cortex (B), corpus callosum (C) and fimbria (D) from P1 to P10. Regional changes in markers associated with TREM2 (red) where represent as color intensity: CD206 (green), CD16/32 (purple), CD86 (blue) and MHCII (orange).