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. 2022 Jul 18;11:e80167. doi: 10.7554/eLife.80167

Figure 8. ASCs and immune cells comprise a dynamic cellular niche.

(A, top) Representative images of fixed frozen mouse cold-exposed iBAT. Tissue was stained with smFISH probes Dcn (green), H2-Ab1 (red), and Top2a (pink). Nuclei were counterstained with DAPI. Images on the right are a magnified view of the boxed regions on the left. Scale bars, 40 μm and 5 μm. (A, bottom) Quantification of cell types within 20 μm around either an H2-Ab1+ cell or a Dcn+ Top2a+ cell (n=3 animals; >100 cells/mouse; mean ± SD). (B) Representative images of fixed frozen mouse cold-exposed iBAT. Tissue was stained with smFISH probes Dcn (green), H2-Ab1 (red), and Nnat (pink). Nuclei were counterstained with DAPI. Images on the right are a magnified view of the boxed regions on the left. Scale bars, 40 μm and 5 μm. (B, bottom) Quantification of cell types within 20 μm around either an H2-Ab1+ cell or a Nnat+ cell (n=3 mice; >100 cells/mouse; mean ± SD). See also Figure 8—source data 1, Figure 8—source data 2.

Figure 8—source data 1. Quantification of the nearest neighbor (20 µM) to either a H2-Ab1+, Dcn+, or Dcn+ Top2a+ cell in the iBAT of cold-exposed mice.
Figure 8—source data 2. Quantification of the nearest neighbor (20 µM) to either a H2-Ab1+, Dcn+, or Nnat+ cell in the iBAT of cold-exposed mice.

Figure 8.

Figure 8—figure supplement 1. Flash-labeling of proliferating cells and proximity of MHCII+ dendritic cells.

Figure 8—figure supplement 1.

(A, top) Procedure for EdU labeling to trace dividing ASCs and differentiated adipocytes. Pdgfra-CreERT2 x LSL-tdTomato were used for genetic tracing of PDGFRA+ cells. Seven days after induction with tamoxifen, animals were housed in the cold for up to 5 days. For dividing ASCs, on the third day, animals were injected with EdU and sacrificed 2 hr later. For differentiated adipocytes, animals were injected with EdU on days 3 and 4 of cold exposure and sacrificed on day 5. (A, bottom) Representative images from iBAT for the experiments outlined, where tissue was stained with EdU (green) or MHCII (pink). tdTomato (red) was detected by native fluorescence. Nuclei were counterstained with DAPI. Scale bar, 10 μm. (B) Alternatively, C57 mice were exposed to cold for up to 5 days. EdU was injected on day three and animals were sacrificed two hours or 2 days after EdU injection. Representative images of iBAT harvested from mice at the indicated times. Tissue was stained with either PDGFRA (red), MHCII (pink), and EdU (green) or PLIN1 (red), MHCII (pink), and EdU (green). Nuclei were counterstained with DAPI.