Sirius red reagent

The slides were dipped in Xylene I and Xylene II, treated with Xylene III, and dipped in ethanol solutions in the following order: 100% to 90% and 80% to 70%. After dipping slides in distilled water thrice, we dipped them in the Harris hematoxylin solution (Sigma, St. Louis cat no: HHS-16, MO, US). Sirius red reagent (Abcam, Cambridge, MA., US) was dropped on the slides and they were incubated. After dipping slides twice in the acid solution, we washed the slides thrice using distilled water, and then dipped them again in ethanol solutions 70% to 80%, 90%, and 100% in 3 steps for each solution. We repeatedly dipped the slides in Xylene I and Xylene II, treated them with Xylene III, and covered the slides with coverslips using permanent mounting media.

Slides were dipped in xylene I, II, and III; xylene was removed using ethanol series (100%, 90%, 80%, and 70%). After the slides were dipped in distilled water three times, they were dipped in Harris hematoxylin solution (Sigma, St. Louis, MO, USA; cat no: HHS-16). Sirius red reagent (Abcam, Cambridge, MA, USA) was dropped onto the slides, and the specimens were incubated. After the slides were dipped twice in acid solution, they were washed three times using distilled water, and then dipped again in the ethanol series. The slides were again dipped in xylene I, II, and III, and covered with coverslips using permanent mounting media.