Picro sirius red stain kit

Lyophilized fibrinogen from human plasma and Millex syringe filter (0.22 μm) were purchased from EMD Millipore (Billerica, MA). Lyophilized thrombin from bovine plasma, calcium chloride, ε-aminocaproic acid (εACA), and Alcian Blue 8GX were purchased from Sigma-Aldrich (St. Louis, MO). Peptides were synthesized by University of Utah DNA/Peptide synthesis core facility. Spectra/Por 7 dialysis membrane (MWCO = 3.5 kDa) was purchased from Spectrum Laboratories (Rancho Dominguez, CA). Coomassie Brilliant Blue R-250 was purchased from Genlantis (San Diego, CA). Sulfosuccinimidyl 6-(3'-(2-pyridyldithio)propionamido)hexanoate (Sulfo-LC-SPDP) and DyLight 680 NHS-ester were purchased from Thermo Fisher Scientific (Newington, NH). Mini-PROTEAN TGX precast electrophoresis gel was purchased from Bio-Rad (Hercules, CA). TO-PRO-3 iodide, Alexa Fluor 488 conjugated anti-rabbit IgG secondary antibody and Alexa Fluor 568 conjugated anti-mouse IgG secondary antibody were purchased from Invitrogen (Carlsband, CA). Rabbit anti-aquaporin 5 (AQP5), rabbit anti-TMEM-16A, rabbit anti-PECAM-1, mouse anti-cytokeratin 7, mouse anti- β-tubulin III, rabbit anti-Ki67 and Picrosirius Red Stain Kit were purchased from Abcam (Cambridge, MA). Mouse Na⁺/K⁺-ATPase antibody was purchased Santa Cruz Biotechnology (Santa Cruz, CA).

Tissue samples from the left lungs, which were fixed with 4% paraformaldehyde were embedded with paraffin wax and subjected to routine processing. Serial paraffin sections (4 μm) were prepared using a rotator microtome, and deparaffinized tissue sections were stained with hematoxylin and eosin (H&E, Sigma-Aldrich, St. Louis, MO, USA) to evaluate morphological changes in the lungs or with Masson’s trichrome stain (Trichrome Stain Kit, Sigma-Aldrich) and picrosirius red stain (Picro-Sirius Red Stain Kit, ab150681, Abcam, Cambridge, UK) to evaluate the collagen content and distribution. Images were obtained using an Axio Imager 2 microscope (Carl Zeiss AG, Oberkochen, Germany) at 400× magnification and Axiocam 506 color (Carl Zeiss AG). Collagen was quantified using ImageJ software (NIH, Bethesda, MD, USA).

The effect of decellularization and/or sterilization on collagen fibers was visualized using the Picro Sirius Red Stain Kit (Connective Tissue Stain; Abcam). Hydrated tissue sections were incubated in Picro Sirius Red solution for 60 min and rinsed with 3% acetic acid and absolute ethanol. Then, tissue sections were dehydrated in absolute ethanol and mounted with Entellan.

Oil Red O staining was performed using a kit from Abcam (ab150678) to analyze lipid deposits in the young and aged human and mouse SKM frozen sections, following the protocol recommended by the manufacturer. Collagens in mouse skeletal muscle were visualized with the Picrosirius Red Stain Kit (Abcam, ab150681) following the manufacturer's protocol.

Lung tissue from the right lower lobe was fixed in 4% paraformaldehyde at 4°C for 24 h, embedded in paraffin, and transversely cut into 4-μm-thick sections. Sections were stained with a Picrosirius Red Stain Kit (Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. Five randomly selected microscopic fields from 3 nonadjacent sections were captured by a light microscope connected to a digital camera. Quantitative assessment of lung collagen with Picrosirius red staining was performed using Image-Pro Plus 5.1 (Media Cybernetics, Rockville, MD, USA), and the results were expressed as a percentage of the total area of the image analyzed as previously described [14 (link)].