Neonatal (P0–7) and adult (1–12mo) mice were sacrificed
using isoflurane, cervical dislocation, and thoracotomy. The heart was quickly
removed, its apex then cut (exposing both left and right ventricles), and washed
in ice-cold PBS to facilitate drainage of blood from the chambers. Hearts were
then fixed in 4% paraformaldehyde (PFA) overnight at 4° C, dehydrated
through a graded ethanol series, cleared in xylene, embedded in paraffin, and
sectioned at 5μm thickness, as previously described15 (link). Movat’s pentachrome staining was
performed using manufacturer’s protocols (American MasterTech). For
Picrosirius Red staining (American Mastertech; Cat. # KTPSRPT), a modified
protocol that excluded Weigert’s Hematoxylin staining was used.
Verhoeff’s staining solution was used to detect elastin (American
MasterTech; Cat. # STVGIGAL). Alizarin Red (Sigma; Cat. # A5533) was used to
determine the presence of calcification. Brightfield images were captured using
the Olympus BX51 microscope retro-fitted with the Nikon DS-Ri1 camera and DS-U3
controller, along with the NIS-Elements BR (version 3.2) software.
using isoflurane, cervical dislocation, and thoracotomy. The heart was quickly
removed, its apex then cut (exposing both left and right ventricles), and washed
in ice-cold PBS to facilitate drainage of blood from the chambers. Hearts were
then fixed in 4% paraformaldehyde (PFA) overnight at 4° C, dehydrated
through a graded ethanol series, cleared in xylene, embedded in paraffin, and
sectioned at 5μm thickness, as previously described15 (link). Movat’s pentachrome staining was
performed using manufacturer’s protocols (American MasterTech). For
Picrosirius Red staining (American Mastertech; Cat. # KTPSRPT), a modified
protocol that excluded Weigert’s Hematoxylin staining was used.
Verhoeff’s staining solution was used to detect elastin (American
MasterTech; Cat. # STVGIGAL). Alizarin Red (Sigma; Cat. # A5533) was used to
determine the presence of calcification. Brightfield images were captured using
the Olympus BX51 microscope retro-fitted with the Nikon DS-Ri1 camera and DS-U3
controller, along with the NIS-Elements BR (version 3.2) software.
