Masson s trichrome stain kit

The kidneys of 3-week-old Zeb2-cKO mice and wild-type littermate controls were dissected and fixed in 4% paraformaldehyde (PFA) or 10% neutral buffered formalin overnight and processed for paraffin embedding following standard protocols. Serial kidney sections were cut and stained using a Periodic Acid Schiff’s (PAS) Stain Kit (24200, Polysciences, Inc.), Picrosirus Red Stain Kit (24901, Polysciences, Inc.), and Masson’s Trichrome Stain Kit (25088, Polysciences, Inc.) according to the manufacturer’s instructions. Slides were examined with an Olympus upright light microscope and photographed using an Olympus DP72 digital camera.

At the study endpoint, hearts were removed from each mouse following PBS perfusion. After a brief PBS wash, individual mouse hearts were embedded in OCT and stored at −80°C until further use. About 8–10-micron serial sections of the aortic root were obtained by cutting the OCT-embedded hearts, as described earlier (27 (link)). Care was taken to ensure that only serial sections collected from aortic root regions representing about 100–150 microns following the valve leaflet were utilized in all morphometric experiments. Additional care was exercised to ensure that staining was performed on sections within similar regions of the aortic root across all treatment groups for quantification and comparison. Aortic root sections were concurrently stained with 0.5% w/v Oil red O (ORO) and hematoxylin and eosin (H&E) to assess the lipid burden and plaque area, respectively. Additional sections were utilized for the detection of collagen content within aortic root lesions using Masson’s Trichrome stain kit (Polysciences). For ORO-stained and MT-stained sections, hematoxylin counterstaining was utilized. All sections were mounted with DPX mounting media, observed using Olympus BX61VS microscope and images were captured using 10× magnification. For quantitative morphometry, eight animals per genotype for each sex with at least 30 sections within each group were analyzed.

Mice skin samples were paraffin-embedded, and sections (5 μm in thickness) were stained with hematoxylin and eosin (HE). Dermal thickness was evaluated by measuring the distance between the epidermal-dermal junction and the dermal-fat junction in HE sections. Skin trichrome staining was performed by Masson’s trichrome stain kit (Polysciences, Warrington, PA, USA). The αSMA positive cells were counted in five random high-power fields using a light microscope. The mean score was used for analysis. The von Willebrand factor (vWF) staining intensity for immunohistochemical assessment was scored semi-quantitatively. The staining intensity (1: negative or weak staining, 2: moderate staining, and 3: strong staining) was evaluated in six randomly selected fields in the subcutaneous area. Then a semi-quantitative score per sample was generated by calculating the average of the six intensity scores per sample.

The Click-iT® EdU Alexa Fluor® 488 Imaging Kit and Alexa Fluor® 594 phalloidin were from Invitrogen. Matrigel (lrECM) and Type I collagen were from BD Bioscience. ShP4HA2 plasmids were purchased from Sigma. 1, 4-DPCA was purchased from Cayman Chemical. Masson’s trichrome stain kit was purchased from Polysciences, Inc. The following antibodies were obtained as indicated: integrin α6 (Millipore); collagen I (Abcam); collagen IV (Abcam); P4HA2 (Santa Cruz); tubulin (Millipore).

To evaluate the collagen deposition in mouse tumor tissues, sections of formalin-fixed paraffin-embedded (FFPE) tissues with a thickness of 5 μm were stained by Masson's trichrome stain kit (Polysciences Inc.) according to the manufacturer's recommendations. Collagen positivity (%) was calculated using automated digital pathology and image analysis software (Visiopharm). On each ROI, background glass and vessel area were excluded from analysis and collagen positivity (%) was automatically calculated as the ratio of the Masson's trichrome–positive area to the entire area (Supplementary Fig. S2).