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. 2021 Aug 20;4:993. doi: 10.1038/s42003-021-02502-6

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a, b IHC for IBA1 reveals macrophage periodicity (arrows) in ducts (a) and ductules (b). c Scatter plot demonstrating that the distributions of inter-macrophage distance differ significantly between lambs aged <2 mo and 5–9.5 mo. Dots represent inter-macrophage distances from 13 individual lambs. Bars represent mean ± standard deviation. **** p < 0.0001. d IHC for IBA1 (brown) and alpha smooth muscle actin (SMA; pink). Arrows indicate macrophages. e, f 3D confocal microscopy of optically cleared ovine mammary tissue with IF for IBA1 (magenta) SMA (cyan) and DNA (DAPI; gold). Images represent 3D maximum intensity projections. Arrow indicates blood vessel. g, h IHC for IBA1 in central (g) and peripheral (h) foci. i Scatter plot demonstrating significantly reduced macrophage abundance in peripheral compared to central foci. Dots represent average macrophage density for individual lambs. * p < 0.05. j–m Serial sections demonstrating IHC for IBA1 (j) Ki67 (k) and CD3 (brown) and CD20 (pink) (m) with accompanying G-O plot for Ki67 (l) (scale (d) parameter = 250 px). Arrow indicates co-localisation of stromal macrophages, a Ki67 hotspot, and a CD3⁺ lymphocyte aggregate. Images are representative of 13 (a, b, g, h), and three (d–f, j–m) biological repeats; mo, months old. All IHC images have haematoxylin counterstain. Scale bar = 40 μm (a, b, d); 100 μm (g, h); 200 μm (j, k, m).