Axioskop 2 mot fluorescence microscope

Adipogenic medium consisted of DMEM/F-12 supplemented with 10% FBS, penicillinstreptomycin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich), 10 µM dexamethasone (Sigma-Aldrich), 66 µM indomethacin (Sigma-Aldrich), 2 µL/mL insulin (insulin lispro injection, 100 U/mL, Humalog ® ) [20] . MSCs were maintained in adipogenic medium for 3 days, detached with 0.05 % trypsin-EDTA, fixed with 4% paraformaldehyde (Sigma-Aldrich) and lipid droplets were stained with fluorescent AdipoRed™ according to the manufacturer's instructions (Lonza). Fluorescent lipid droplets were detected with Axioskop 2 Mot fluorescence microscope (Carl Zeiss) using EC Plan-Neofluar 20x/0.5 objective (Carl Zeiss). For quantitation of lipid accumulation in adipocytes, the cells (5 × 10 5 cells/sample) were labeled with AdipoRed™ as described above and quantified by FACSCalibur flow cytometer using CellQuest TM software.
Osteogenic medium was composed of CM and 10 mM β-glycerophosphate (Sigma-Aldrich), 50 µg/mL L-ascorbic acid 2-phosphate (Sigma-Aldrich) and 0.1 µM hydrocortisone (Sigma-Aldrich). MSCs were cultured in osteogenic medium for 14 days then fixed with 8% formaldehyde and stained with Alizarin Red S (Sigma-Aldrich) solution (pH 4.1) to detect calcium deposition. Photomicrographs were taken with Olympus CKX41 inverted light microscope and Olympus Camedia C-5060 camera (Olympus Holding Europa GmbH).

The 4 μM sections were dewaxed twice in xylene for 5 mins each time, followed by rehydration using gradient alcohol (100%, 90%, 80%, 70%) for 3 mins each. After washing the slide once with PBS, the tissue was circled using a waterproof pen. For immunofluorescence staining, Antigen repair solution was applied to the tissue at a temperature above 95˚C for 10 mins. The tissue was then blocked with 10% goat serum for 2 hours, followed by incubation with the anti-Cryptosporidium antibody Sporo-Glo (Waterborne) at a ratio of 1:50 in 0.1% BSA-PBS for 1 hour. After rinsing with PBS, the slices were treated with the Slowfade Gold anti-fading agent containing DAPI reagent (Invitrogen) and covered with glass slides. For PAS staining, after rehydration and washes with ddH 2 O, the samples were incubated in periodic acid (Shyuanye, Shanghai, China) for 5 mins, washed 3 times with ddH 2 O, incubated in Schiff's reagent (Shyuanye, Shanghai, China) for 15 mins and washed 10 mins with tap water. Then, the rest of the staining was followed by the steps of H&E staining described above. The stained slides were examined under a Zeiss Axioskop Mot 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany), and the C. parvum counts were determined using ImageJ software.

The C57BL/6j mice were subsequently euthanized and the ileum samples were collected at 3 dpi, 11 dpi and 30 dpi, with 4 mice in each group per time point. The collected samples were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and subsequently cut into 4 μm-thick sections. Hematoxylin and eosin staining was performed using conventional procedures by the Servicebio company. To evaluate the function of ileum mucosal barrier in mice, we measured the length of villi, depth of crypts, and crypt depth ratios. Additionally, we counted the number of parasites by observing microscopic parasites per 15 intestinal villi at three different infection periods. The stained slides were examined under a Zeiss Axioskop Mot 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany), and analyzed using Ima-geJ software. Histological pathology was evaluated based on the criteria used in previous study: crypto damage (0-4 score), severity of inflammation (0-3 score), and depth of injury (0-3 score) [18] (link).

The 4 μM sections were obtained using a cryostat microtome (Leica, Wetzlar, Germany) and placed onto glass slides. The paraffin sections underwent two rounds of dewaxing in xylene, followed by gradient alcohol treatment (100%, 90%, 80%, 70%) for 3 mins each. The sections were then washed with PBS and repaired with acid antigen repair solution at 100˚C for 10 mins. The sections were treated with 10% goat serum at room temperature for 1 hour, then incubated with E-cadherin (Abmart, Berkeley Heights, NJ, USA) diluted in 1:100 for overnight at 4˚C. Subsequently, the slices were incubated with Alexa Fluor 488 coupled goat anti-mouse IgG (ThermoFisher) diluted in 1:1000 and Sporo-Glo diluted in 1:50 at room temperature for 1 hour. The slides were rinsed with PBS and then treated with the Slowfade Gold anti-fading agent containing DAPI reagent (ThermoFisher) to the cover glass. The stained slides were examined under a Zeiss Axioskop Mot 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). The images were processed using ZEN microscopy software (Carl Zeiss, Oberkochen, Germany).