H3136

The collected lung and ileal tissues were rinsed with PBS, followed by fixation and dehydration using 4% paraformaldehyde and a series of ethanol, respectively. Thereafter, the tissues were embedded in paraffin and cut into sections (5 μm thick) for staining with hematoxylin (H3136, Sigma, USA) and eosin (HE, E4009, Sigma, USA). The histopathological changes in the lung and ileal tissues were observed using a microscope (Eclipse Ci-L, Nikon, Japan).

To evaluate the protein expression, immunocytochemistry (ICC) technique was applied. After 5 days of coculture, the islets in all groups were fixed in 4% paraformaldehyde (Gibco, Germany) and embedded in low melt agar (Sigma, Germany). In the direct cocultured group, both islet and MSCs were fixed and separated from the plate with a scraper. After preparing 5 μm sections and deparaffinization, the slides were subjected to ICC using primary antibodies against Bcl-2 (Abcam, France, #ab115807), Bax (Abcam, France, #ab69643), active caspase-3 (Abcam, France, #ab32042), HIF-1α (Medaysis, USA, #RM0374), and p53 (Dako, USA, #M7001). HRP-secondary antibody (Abcam, France, #ab6717) was used for detection after overnight incubation with primary antibodies. Positive cells for protein expression appeared with 3,3′-diaminobenzidine (Sigma, Germany, #D12384) staining and counterstained by hematoxylin (Sigma, Germany, #H3136). The protein expression rate was calculated by the H-score method using the following formula: H − score = 1 × (%mild staining) + 2 × (%moderate staining) + 3 × (%strong staining) [3 (link), 28 (link)].

The lungs were dissected upon culling and fixed in 4% paraformaldehyde for at least 48 h, before being transferred to PBS. The lungs were placed into a tissue cassette and embedded in paraffin wax, and 5-μm sections were generated and transferred to microscope slides using a microtome. Sections were stained with hematoxylin (Sigma, H3136) and eosin (Sigma, HT110116) (H&E), by moving slides sequentially through a series of reagents, spending 1–2 min in each reagent: xylene, 100% ethanol, 90% ethanol, water, hematoxylin, water, 95% ethanol, eosin, 95% ethanol, 100% ethanol, and xylene.
Histology slides were analyzed on a Nikon Eclipse E600 using either a ×10 or ×20 objective lens, and images were obtained using the NIS-Elements software. Alveoli were identified by an airspace (lumen) surrounded by characteristically thin squamous epithelial cells (pneumocytes). These were distinguishable from bronchioles, which have a thicker wall of oblong epithelial cells. Within the NIS-Elements, the polygon tool was used to manually trace the edges of individual alveoli. This tool generates a perimeter and area measurement for each shape, which were exported for analysis. Alveoli were classified as intact or damaged. Twenty alveoli per mouse, across two sections of lung tissue, were measured for analysis.

For observing the histopathological changes in nasal mucosa after CGA or Dex intervention, a 4-cm-thick mouse nasal mucosa was fixed in 4% paraformaldehyde (P6148, Sigma–Aldrich, U.S.A.), dehydrated by ethanol (E7023, Sigma–Aldrich, U.S.A.), blocked in paraffin (327204, Sigma–Aldrich, U.S.A.), and sliced. Then, the sliced mouse nasal mucosa was soaked in xylene (95670, Sigma–Aldrich, U.S.A.) for dewaxation. Gradient ethanol 100, 90, 80, and 70% was used to rehydrate mouse nasal mucosa slices. Mouse nasal mucosa slices were stained by Hematoxylin (H3136, Sigma–Aldrich, U.S.A.) for 15 min, differentiated by being soaked in 5% acetic acid (A6283, Sigma–Aldrich, U.S.A.), and immersed in distilled water for developing blue. Next, mouse nasal mucosa slices were stained by Eosin (E4009, Sigma–Aldrich, U.S.A.) for 10 min. Gradient ethanol 70, 80, 90, and 100% was used to dehydrate mouse nasal mucosa slices. After soaking in xylene twice, mouse nasal mucosa slices were sealed by Canada balsam (60610, Sigma–Aldrich, U.S.A.) and observed using an optical microscope (M3800, Fisher Scientific, Waltham, MA, U.S.A.).

Sample slides were stained with haematoxylin (H3136, Sigma) for 5 minutes; colour was checked under the microscope. Slides were then washed with tap water 3 times, followed by bluing in Scott's tap water (S5134, Sigma) for 30 seconds. After another 3 times of washing with tap water, slides were counterstained with Eosin Y (230 251, Sigma) for 40 seconds, followed by washing with tap water for another 3 times. After the final wash, slides were mounted with mountant (06 522, Sigma), covered with coverslip, dried overnight and checked with a microscope connected with computer using Olympus cellSens software (Version 2.1).

Paraffin sections of rat hippocampal and renal tissues were prepared. After treatment of the sections with xylene and gradient ethanol, HE staining (Sigma, H3136; E4009) was performed. The sections were adequately stained, dehydrated, transparently sealed, and microscopically examined. The semi-quantitative score of renal tissue is according to the Paller and coworkers report [32 (link)], which is generally based on renal tubular injury including tubular lumen obstruction, brush border loss, cytoplasmic vacuolization etc. Furthermore, evaluation of hippocampal tissue is conducted through necrotic neuron quantification in the CA1, CA3, and Dentate gyrus (DG) regions, as well as assessment of neural cell arrangement [14 (link)].