Bx50 microscope

After mice were sacrificed on day 56, lungs were fixed with 10% formalin infusion through a tracheal cannula at a constant pressure of 25 cm H₂O. The lungs were immersed in fixative for at least 24 h, after which the left lung was embedded in paraffin. After paraffin embedding, 5 μm sections were cut and de paraffin sections were first deparaffinised. Endogenous peroxidase activity was blocked with 0.3% H₂O₂ (Merck) in methanol for 30 min at room temperature and rehydrated in a graded ethanol series to PBS and paraffin section were stained with hematoxylin/eosin (H&E) for inflammation, periodic acid-schiff (PAS) for goblet cells, Masson’s trichrome for connective tissue, rabbit polyclonal anti-α-smooth muscle actin antibody (Abcam, Cambridge, UK) for smooth muscle cells and rabbit polyclonal anti-Ki76 antibody (Abcam) for proliferating cells according to standard methods. Photomicrographs were taken with an Olympus BX50 microscope equipped with a Leica DFC 320 digital Camera.
Slides were reviewed in blinded fashion by two observers independently and slides were scored on the basis of the percentage of positive stained cells in the following way: -, no positive staining; +/-, less than 25% of cells stained positive; +, 25 to 50% cells stained positive; ++, 50 to 75% cells stained positive.

After fixation in 10% formalin, jejunum samples were embedded in paraffin. Paraffin sections (5 µm) were deparaffinized, and endogenous peroxidase activity was blocked with 0.3% H₂O₂ (Merck, Darmstadt, Germany) in methanol (30 min) and rehydrated in a graded ethanol series to PBS. After antigen retrieval in 10 mM citrate buffer (PH 6.0) for 10 min in a microwave, sections were pre-treated with 5% goat serum (Dako, Glostrup, Denmark) before overnight incubation with rabbit-polyclonal Ki67 antibody (1:1000, ab66155, Abcam, Cambridge, UK) or caspase-3 (1:1000, Cell Signaling, Danvers, MA, USA) at 4 °C. Tissue sections were sequentially incubated with biotinylated goat-anti rabbit (1:200, Dako, Glostrup, Denmark) followed by streptavidin-biotin complex/horseradish peroxidase (Vectastain Elite ABC, Vector Laboratories, Peterborough, UK). Staining was visualized using 0.05% diaminobenzidine (DAB) solution for 10 min, and sections were counterstained with Mayers’ hematoxylin (Merck Millipore, Amsterdam, The Netherlands). Photomicrographs were taken with an Olympus BX50 microscope equipped with a Leica 320 digital camera.

As previously reported (Perez-Pardo et al., 2017 (link)), coronal brain slices of 40 μm were sectioned using a cryostat (CM3050, Leica Microsystems). After incubation with 0.3% H₂O₂ for 30 min and following blocking serum, sections were incubated overnight with rabbit anti-tyrosine hydroxylase (TH; Santa-Cruz Biotechnology, 1:1,000). Next day, sections were incubated with a biotinylated secondary antibody (Jackson ImmunoResearch, 1:200) for 2 h. The avidin-biotin method was used to amplify the signal (ABC Kit, Vector) and staining was visualized using 0.05% DAB solution. Digital images of immunostained sections were captured with an Olympus BX50 microscope equipped with a Leica DFC 320 digital camera. TH-immunopositive neurons were quantified stereologically on regular spaced sections. Analyses were performed by researchers that were blind to the treatment condition of the sample.

A slice of the liver tissue from the left lobule was fixed in 10% neutral phosphate-buffered formalin for at least 24 h and then embedded in paraffin after the tissue-processing process had been done according to the standard protocol provided for laboratory animals [16 (link)]. Cross-sections of the liver tissue were made on a Leica microtome (Leica Microsystems, Germany) with a thickness of 5 μm, and then deparaffinized, hydrated, and stained with hematoxylin and eosin (H&E) protocol. Pathologists have analyzed in great detail the changes (the presence of necrosis, hyperplasia, and hypertrophy of Kupffer cells, vacuolar, hydropic, and fatty hepatocytes, as well as inflammatory infiltrate) in all three zones of the liver lobule for each animal individually in each group. The morphometric analysis was used to determine the extent of the necrotic area on a selected H&E field. For each animal in each group, 10 microscopic fields at 200× magnification were examined by morphometric analysis. An Olympus BX-50 microscope (Shinjuku, Tokyo, Japan) with a Leica digital camera (DFC295, Reuil-Malmaison, France) was used to obtain photomicrographs.

For histomorphometric analysis, ileum samples collected from vaccinated mice were fixed in 10% neutral buffered formalin in the form of Swiss rolls and embedded in paraffin. The 5-μm sections were stained with hematoxylin/eosin according to standard procedures [41 (link)]. Photomicrographs were taken with an Olympus BX50 microscope equipped with a Leica DFC 320 digital camera (magnification of 200×). The morphometric analysis of the sections was performed on 10 randomly selected, well-oriented villi and crypts per animal. A computerized microscope-based image analyzer (Image Pro MC) was used to determine villus height (measured from the tip of the villus to the villus–crypt junction) The villus height was manually defined for each villus separately [12 (link)].