Masson s trichrome stain kit

All rats were euthanized on day 10. The wound area and surrounding normal tissue were harvested and cut into halves; one piece was immediately fixed in 4% paraformaldehyde solution for section staining, and the other was frozen in liquid nitrogen for Western blot analysis. After fixation, the tissues were embedded in paraffin. The sections (5 μm) were stained with hematoxylin and eosin (H&E) (Merck Millipore, Darmstadt, Germany) and Masson’s trichrome stain kit (ScyTek Laboratories, Logan, UT, USA), following the manufacturer’s protocol. The stained sections were viewed and photographed using an Axio Scan Z1 slide scanner (ZEISS, Oberkochen, Germany).

The heart sections were also stained with Masson’s trichrome stain kit (ScyTek Laboratories, Inc., UT, USA) as previously described [25 (link)]. Brief, the heart sections were incubated with Bouin solution at 60°C for 45 min, washed in running tap water until the yellow color disappeared. Then each slide was immersed in Weigert’s Haematoxylin solution for 5 min, washed in running tap water. After that, each slide was stained for 15 min with Acid Fuchsin, rinsed in distilled water. The slides were incubated for 10 min with a phosphomolybdic acid solution and then immediately stained for 10 min with methyl blue solution, rinsed in distilled water. Then slides were treated with 1% acetic acid solution for 3 min or until bluish collagen fibers were observed. Each slide was dehydrated through two changes of 95% alcohol followed by two changes of 100% alcohol, and then placed in xylene. The quantification of fibrotic areas (stained blue) and myocardial areas (stained red) was measured by ImageJ analysis software. The fibrosis percentage was calculated by ratio of fibrotic area to the myocardial area.

The paraffin-embedded skin specimens were measured using Masson’s trichrome stain Kit (ScyTek Laboratories, Inc., UT, USA). The slides were stained with Bouin’s Fluid and Weigert’s iron hematoxylin working solution. Furthermore, the slides were differentiated in phosphomolybdic-phosphotungstic acid solution and stained with aniline blue solution. Finally, the stained skin specimens were dehydrated in series. The slides were examined microscopically and the images were recorded.

For immunohistochemical analysis, kidney tissues from the sacrificed mice were harvested and fixed in 10% formaldehyde. Paraffin cross-sections with a thickness of 5 μm were stained with antibodies against MT-I/II (Invitrogen), followed by N-Histofine^® MOUSESTAIN KIT (414321F; Nichirei Biosciences Inc.; Tokyo, Japan). To measure renal fibrosis, the paraffin-embedded sections were stained using Masson’s Trichrome stain kit (ScyTek Laboratories Inc., Logan, USA). Quantification of kidney immunohistochemical assay and fibrosis on Masson’s trichrome-stained sections were performed using ImageJ analysis. The raw pixel values of the selected areas were estimated using the ImageJ software. These values indicated the distribution area times the stain intensity of the images.