Formaline

After imaging, the animals were euthanized with an overdose of pentobarbital sodium (250 mg/kg i.p.) and their lungs were collected for histopathological examination. Lungs were perfused through the interventricular septum with phosphate-buffered saline (Sigma Aldrich, Sweden) and then inflated with formaline (neutral buffered 10%, Sigma Aldrich, Sweden) via the trachea using gravity inflation at 25 cm H₂O. The trachea was tied and the inflated lungs removed and placed in formalin for 24 hours before continued processing and embedding in paraffin. Two 3 μm sections, comprising both the left and the right lobes, were cut at the level of the tracheo-bronchial tree and stained with hematoxylin and eosin (H&E) before microscope examination.

Acetonitrile, methanol, ortho-phosphoric acid (HPLC grade), rutin, hyperoside, hypericin and hyperforin were purchased from Sigma-Aldrich, Germany for phytochemical profiling. Quercetin dihydrate (≥98%) was obtained from Carl Roth (Karlsruhe, Germany). Hydroxyethyl cellulose (HEC), hydroxy propylmethyl cellulose (HPMC), sodium carboxymethyl cellulose (NaCMC), and Pluronic F-127 were purchased from Sigma-Aldrich, Germany for the formulations. Ketamine HCl 5% (Rotex medica, Trittau, Germany), and Panthenol® 2% cream (Nile Co. for Pharmaceuticals and Chemical Industries, Egypt) were obtained for the in vivo experiments. Formaline, xylene, hematoxylin and eosin stain, and Masson’s trichrome stain were sourced from Sigma-Aldrich, Germany for the histopathology. All other chemicals and reagents were of analytical grade.

Before immunofluorescence staining, the cells were washed from the conditioned medium with phosphate-buffered saline (PBS) and fixed in a solution containing 10% formaline (Sigma-Aldrich, Cat. No. HT5011) for 20 min at room temperature. Then a series of PBS washes were performed and the cell membranes were permeabilized with a 0.25% Triton X-100 solution in PBS for 5 min at room temperature. Then the cells were washed with PBS 3 times and incubated in a blocking solution containing 5% bovine serum albumin (Sigma-Aldrich, Cat. No. A9403) diluted in PBS for 1 h at room temperature to prevent nonspecific antibody binding. Then the cells were incubated with primary antibodies diluted in a blocking solution at a concentration indicated in Table 1 overnight at a temperature of +4 °C. Then a series of washes were performed with a PBS solution, after which the cells were incubated with secondary antibodies for 1 h at room temperature, diluted in blocking solution to the concentration indicated in Table 1. Then a series of washes were performed with a PBS solution and stained with DAPI, after which the cells were fixed on a slide using Mounting Medium with DAPI (Cell Signalling, Danvers, MA, USA, Cat. No. 8961S). Images were captured with Olympus Fluoview FV3000 laser confocal system.