Fig. 2: Histological changes in Brn-3b KO aorta compared with WT.

a Representative images of intact aortas taken from WT or Brn-3b KO mice, after staining for oil red. Images shown at X5 magnification with boxed areas expanded to highlight narrowing/constriction in Brn-3b KO aortas and corresponding WT aortas. b H&E staining of longitudinal aortic sections from WT (i) or Brn-3b KO mice (ii). 20× magnification of selected areas shows increased adventitial thickness highlighted by dotted lines (arrows indicate specific areas of thickening in KO aortas; * highlights increased WAT deposition) in Brn-3b KO but not WT aortic sections. L lumen. Images (b–g) were captured using Hammamatsu Nanozoomer imaging system and shown at ×5; ×20 and ×40 magnification. TI tunica intima, TM tunica media, TA tunica adventitia, WAT white adipose tissue, PVAT perivascular adipose tissue. c Representative images showing Masson’s trichrome staining of longitudinal aortic sections from either (i) WT or (ii) Brn-3b KO mice which highlights the increases in thickness of tunica adventitia (TA) layer and elastin disruption in tunica media (TM) of Brn-3b KO aortas (*), when compared with WT controls. Red staining represents cytoplasm; dark purple/black shows cell nuclei; blue staining indicates ECM protein (e.g. collagen) deposits. d Representative images showing immunostaining for collagen protein, Col1a1, in WT and Brn-3b KO aorta sections using DAB immunostaining protocol, with intense brown staining indicating increased expression in aortic sections from Brn-3b KO mice compared with WT controls. e (i) Quantification of TA width thickness in representative aortic sections, with each point representing the mean values from eight independent mice within each group. Data represents mean and standard deviation (±SD) with significance (***p < 0.001), determined by students t test. (ii) Graph showing differences in aortic lumen diameter from WT or Brn-3b mice (n = 8 independent aortas/group). Analysis done using students t-test. f Masson’s trichrome stained aortic section highlighting disruption of elastin fibres in Brn-3b KO mutants; indicated by fragmented blue staining (*), compared with WT aortas. g van Gieson staining of elastin fibres (dark brown) in aortic section from WT or Brn-3b KO mice showing disruption of elastin fibres in Brn-3b KO aortas (arrowheads) when compared with WT aortas. Magnification is shown at 5× (top panels) and 40× magnification (bottom). h–j Transmission electron microscopy images showing differences between aortas taken from WT and Brn-3b KO mice. h Increased collagen fibres in the Tunica adventitial (TA) layer with Brn-3b KO aortas showing significantly thicker TA with more compact arrangement of collagen fibres when compared with WT controls. i Representative images of the tunica media (TA) showing elastin fibres and VSMCs found between the elastic fibres (E) are indicated. Disruption of elastin fibres in Brn-3b KO aortas are indicated by yellow arrows, when compared with WT aortas. j Images showing aortic perivascular adipose tissue (PVAT) surrounding the aorta taken from either WT or Brn-3b KO mice. Black arrows indicate larger numbers of infiltrating lymphocytes in Brn-3b KO PVAT when compared with WT controls.