Weigert s iron hematoxylin solution

Scaffold-tissue constructs for histological analysis were cut opened along the height axis. Samples stained for GAGs were first stained for 10 min in Weigert's iron hematoxylin solution (Sigma-Aldrich) to counterstain the cell nucleus black, washed thoroughly in running tap water and stained for 3 min with Fast green solution (Sigma-Aldrich), and quickly dipped in 0.5% acetic acid. Finally, samples were stained for 5 min in a 0.1% solution of Safranin-O (Sigma-Aldrich), after which they were rinsed with PBS until the solution was clear. Picrosirius red (Abcam) staining was performed following the manufactures’ instructions. In brief, samples were incubated for 2 h in Picrosirius red solution and then rinsed quickly in 0.5% acetic acid solution (both from Abcam, Cambridge, UK). Afterward, samples were rinsed in PBS 3 times.

Mice were sacrificed on day 37. The ankle joints were collected, fixed in 4% paraformaldehyde for 48 h, decalcified in 10% ethylenediaminetetraacetic acid solution, and embedded in paraffin. The tissue sections were stained with hematoxylin and eosin (HE), Weigert’s Iron Hematoxylin solution (Sigma-Aldrich) and Fast Green solution (Sigma-Aldrich) for histopathological analysis. The MPO and NE expression levels were examined using immunohistochemistry with anti-NE and anti-MPO antibodies in accordance with the instructions of the manufacturer.

After tissue recovery, aortic tissue was marked with a methylene blue pen to ensure accurate orientation (circumferential vs longitudinal) of the tissue. The tissue was then fixed in 10% formalin and embedded in paraffin. Aortic samples were stained with Accustain Elastin Verhoeff's Van Gieson kit (Sigma Aldrich, St. Louis, Mo) according to the manufacturer's instructions. Masson trichrome staining was performed with Bouin's solution and Weigert's iron hematoxylin solution (Sigma Aldrich, St. Louis, Mo). Representative sections both circumferentially and longitudinally were reviewed by a pathologist blinded to the underlying disease process (control, chronic dissection, or aneurysm). Specimens were graded qualitatively from 0 to 3 on mucoid extracellular matrix accumulation (MEMA), elastin fragmentation, medial fibrosis, and medionecrosis.²⁵ (link)

After deparaffinization, the left knee joint was treated with Weigert’s Iron Hematoxylin solution (Sigma, USA) for 10 min and stained with 0.001% Fast Green solution (Sigma, USA) for 5 min. The sample was then incubated with 1% acetate solution for 10 s and stained with 0.1% safranin O solution (Sigma, USA) for 5 min. Thereafter, the tissue was dehydrated and observed under an optical microscope (Nikon, Japan).

Livers were fixed with a 10% formaldehyde solution and embedded in paraffin. Histology microscopic evaluation was performed in 10-μm thick sections of this tissue previously deparaffinized and stained. For this purpose, Mayers hematoxylin (Sigma-Aldrich, United States) and eosin (Leica, Germany) and conversely, Weigert’s iron hematoxylin solution and picrosirius red (Sirius Red F3BA 0.1% wt/vol in saturated picric acid; Sigma-Aldrich, United States) washed in glacial acetic acid (Probus, Madrid, Spain) and water (5:1,000) were used, followed by dehydration with ethanol (VWR, Radnor, PA, United States) and mounting in Entellan (Merck, Germany).

Van Gieson’s staining solution, i.e., 0.1% picro-fuchsin solution, was used for assessing the collagen. It was made by mixing 1% acid fuchsin aqueous solution (acid fuchsin powder: Thermo Scientific, Waltham, MA, USA, #400210250) with 1.2% picric acid aqueous solution (RICCA Chemical, Arlington, TX, USA, #5860-16) at 1:9 ratio. Weigert′s iron hematoxylin solution (Sigma-Aldrich, St. Louis, MO, USA, #HT1079-1SET) was used to stain nuclei. For immunofluorescence staining, antigen retrieval was performed with 10mM citrate buffer with 0.05% Tween-20, pH 6.0. Sections were stained with the following primary antibodies: UCHL1/PGP9.5 Polyclonal antibody (1:300, Proteintech, Rosemont, IL, USA, #14730-1-AP, gift from Dr. Brian Lin, Tufts University) and tryptase antibody (1:100, Abcam, Waltham, MA, USA, #ab151757). Secondary antibodies are the Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Invitrogen #A-11008) and the Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (Invitrogen, Waltham, MA, USA, #A-11012). DAPI was used to counterstain sections. Sections incubated with only one primary antibody served as single-positive controls. Sections incubated with secondary antibodies only served as negative controls.