Photomicroscope

At the end of the studies animals were sacrificed while they were under isoflurane (5% in 100% oxygen) anaesthesia. For histological examination, paw tissues were taken 6 h after edema was induced by carrageenan. The tissue slices were immediately fixed in freshly prepared 10% neutral buffered formalin for a minimum of 24 h. On the other hand, specimens of the gastric walls from each rat were kept in 10% buffered formalin for 24 h for histopathological examination following the assessment of ulcer score for anti-ulcer activity. Then the tissue specimens were processed for paraffin embedding tissue sections. The samples were sectioned with a microtome, stained with hematoxyline and Eosin (H and E) and mounted on Canada balsam. All sections were examined under light microscope. Photographs of the lesions were taken with an Olympus photo microscope for observation and documentation of histopathological changes such as oedema, inflammation, infiltration and erosion.

For observations of the number of fluorescent cells under phase-contrast and fluorescence microscopy, all B. thuringiensis strains were cultivated in LB liquid medium at 28°C. The morphology of the strains was observed with an Olympus photomicroscope (Tokyo, Japan).
To observe the production of parasporal crystals under phase-contrast microscopy, all strains were sporulated using a previously described method [37 (link)]. Briefly, strains were cultured in ICPM liquid medium (0.6 % peptone, 0.5 % glucose, 0.1 % CaCO₃, 0.05 % MgSO₄, and 0.05 % KH₂PO₄ (pH 7.0)) for 36 h at 28°C. The spores and crystals were collected and washed three times with 1 M NaCl solution and three times with water [37 (link)]. These samples were also used to detect crystal protein expression by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Under phase-contrast microscopy, spores are phase-bright, and crystals are phase-dark [35 (link)]. For scanning electron microscopy with a Quanta200 (FEI, Hillsboro, OR, USA), the lysed cell samples were treated following the methods described by Shao et al. [37 (link)].

Excised lungs were fixed with 4% formaldehyde for 48h, and embedded in paraffin for slide preparation. Before staining, 5-μm slides were dewaxed by xylene and rehydrated by a graded series of ethanol. The sections were stained by hematoxylin and eosin (HE) to evaluate pathogenesis. The procedure of IHC was described in the previous study [40 (link), 41 (link)]. Ten % BSA-blocked slides were incubated with the primary antibody targeting IL10 (Bioss Inc., Woburn, MA, USA), CD163 (abcam^®, Cambridge, MA, USA), EGFR (Cell Signaling Technology, Beverly, MA, USA) and p-Src (Y419; GeneTex, Inc., Irvine, CA, USA) for 1h, at room temperature. Subsequent procedures were performed using Vectastain ABC kit (Vector Laboratories, Burlingame, CA) according to the instruction, and photographed by photomicroscope (Olympus, Melville, NY, USA).