Masson goldner staining kit

Masson’s trichrome stain (Goldner variant) was used in both ex vivo studies to detect collagen and general tissue morphology.
Skin explants were fixed in 10% buffered formalin for 48 hours and processed overnight in increasing concentrations of ethanol, followed by three washes with xylene and subsequent equilibration in paraffin. Tissues were then embedded in paraffin blocks and cut into 5 µm sections. For staining, sections were deparaffinized, washed with xylene, and rehydrated for the preparation of Masson–Goldner or Alcian blue staining. For Masson–Goldner, the Masson–Goldner staining kit (1.00485; Sigma-Aldrich Co.) was used as per the manufacturer’s instructions. In the hydrocortisone study, Alcian blue was used to stain proteoglycans. For this, samples were incubated with Alcian blue (8GX, A3157; Sigma-Aldrich Co.) for 30 minutes and rinsed with deionized water. Finally, slides were washed with 1% acetic acid and xylene and mounted.

The histological samples, after decalcification process (Osteosoft®) were dehydrated in alcohol series and embedded in paraffin. Sections 5 μm thick were stained with haematoxylin and eosin and Masson–Goldner trichrome (Masson-Goldner staining kit, Merck). Material was analysed in light microscope (80i Eclipse, Nikon).
Microscopic analysis of samples A and B were evaluated using EVO LS15 ZEISS Scanning Electron Microscopy (Zeiss, Jena, Germany). The tested samples were dehydrated in a series of ethanol solutions, dried and sputtered with gold.^{27 (link)}

Tissues that were dewaxed and rehydrated in a descending alcohol series were incubated with Weigert's iron hematoxylin nuclear staining solution for 5 min (Merck Millipore, Darmstadt, Germany). After 5 min of flushing in tap water, slides were submerged in 1% acetic acid for 3 min. Subsequently, the sections were immersed respectively in Azoploxine solution for cytoplasm dyeing (5 min), Tungstophosphoric acid orange G solution for erythrocytes staining (1 min), and Light green SF solution for collagen and connective tissue visualization (5 min). The slides were washed in 1% acetic acid between each staining procedure for 3 min (Masson-Goldner staining kit, Merck Millipore, Darmstadt, Germany). In the last step, slides were washed in 1% acetic acid for an additional 5 min and left to dry. Afterwards histological slides were covered with non-aqueous mounting agent (Neo-Mount anhydrous mounting medium; Merck KGaA, Germany) and a cover glass for further analysis.
Microscopic examination and photographs were made using a bright field microscope (Olympus CX41) connected with camera equipped with Olympus Stream Image Analysis Software (Olympus Europe Holding GmBH). Blinded microscopic examination was performed at 2 magnifications (×10 and ×40) and described according to the scoring described in Table II and figure legends.

Masson staining was performed with a Masson-Goldner staining kit (Merck KGaA), following the manufacturer's protocol, to detect collagen deposition in liver tissues. Briefly, rehydrated sections (5 µm) were staining with Weigert's iron hematoxylin staining solution for 5 min, followed by staining with Azophloxine solution for 10 min, Tungstophosphoric acid orange G solution for 1 min and Light green SF solution for 2 min. Washes with 1% acetic acid were performed between the staining steps. Following dehydration in ethyl alcohol, the sections were mounted. All the procedures were performed on room temperature. The images (magnification, ×200) were obtained by an Axio Vert.A1 inverted microscope (Zeiss AG, Oberkochen, Germany).