Inverted bright field microscope

UCB-CD34⁺ cells, non-cultured and cultured for 10 days, were plated in a 24-well plate at 150 cell/well in cytokine-supplemented methylcellulose medium (MethoCult H4435, StemCell Technologies, Canada) and further cultured for 14 days at 37°C and 5% CO₂. Colony forming unit colonies were counted using an inverted bright field microscope (Leica, Germany).

Juvenile (P21) and adult (P120–210) mice were overdosed with ketamine/xylosine and transcardially perfused with ice-cold PBS and 4% PFA. Spinal cords were post-fixed in 4% PFA overnight, cryoprotected in 30% sucrose and frozen in OCT compound on dry ice. Lumbar spinal cords were sectioned at 30–40 µm at levels L3–L6 and stored in PBS. Free-floating immunohistochemistry was performed on 5–10 spinal cord sections per mouse. Sections were incubated overnight in primary antibody solution (Goat anti-ChAT, Millipore 1:100, 1.5% NDS in PBST-x) at 4°C on a rocker. After three washes in PBST-x sections were incubated in biotinylated donkey anti-goat secondary antibody in PBST-x (1:500, Jackson Immunoresearch) for 1 h. Followed by three washes in PBST-x, sections were incubated in ABC Vectashield kit solution (Vector, as per manufacturer's instructions) for 1 h. Antibody binding was visualized using diaminobenzidine kit (Vector Labs, as per manufacturer's instructions). Sections were mounted on glass slides, dehydrated in series of ethanol solutions and imaged using a Leica inverted bright field microscope.

Cellular senescence was assessed using a β-Galactosidase Staining Kit (Beyotime). Briefly, culture media was removed and cells were washed with PBS, cells were fixed with 4% paraformaldehyde (PFA, v/v) at RT for 15 min. Fixation solution was removed and cells were rinsed 3 times with PBS for 5 min each. Cells were then treated with 1 mL staining solution and incubated overnight at 37 °C. β-Galactosidase staining was then observed using a bright field inverted microscope (Leica).

Lungs were catheterised, perfused with 10% neutral buffered formalin and sutured shut to maintain lungs in an inflated state. They were submerged in 10% formalin for 24 hours followed by 24 hours of 100% ethanol and 24 hours of 70% ethanol. Lungs were then processed using an Excelsior AS tissue processor and embedded in paraffin wax. Haematoxylin and eosin (H&E) staining was carried out using 6‐μm tissue sections according to standard procedures. Briefly, sections were deparaffinised with xylene, rehydrated in decreasing percentages of alcohol and washed with water. Rehydrated sections were stained in Mayer's haematoxylin solution for 6 minutes. Sections were washed in running tap water for 4 minutes to undergo “blueing”. Sections were counterstained with eosin for 2 minutes and rinsed in the water bath. Slides were dehydrated using a graded alcohol series ending with two changes of absolute alcohol. Slides were then cleared with xylene. Xylene was evaporated and sections were covered with Histomount xylene‐based mounting solution and slides were left in a 37°C oven overnight to set. Images were captured using a Leica bright‐field inverted microscope. Metastatic lesions were detected and each section was given a metastatic score: 1 = no visible metastatic lesion, 2 = 1 metastatic lesion, 3 = 2 metastatic lesions and 4 = 3 or more metastatic lesions.

After 14 days of adipogenic differentiation, phase contrast images were captured using an inverted brightfield microscope (Leica Microsystem, Germany). Cells were then fixed for 20 min with 4% paraformaldehyde, stained for 15 min with oil red O solution (oil red O 0.5% in isopropanol, diluted 3:2 in deionised water) at room temperature and images were acquired using an inverted microscope (Leica Microsystems, Germany). For semi-quantitative analysis of oil red O staining, the dye was extracted with 100% isopropanol, the solution was centrifuged at 500 g for 2 min, and absorbance was measured at 520 nm using a Varioskan Flash plate reader (ThermoFisher Scientific).

After 14 days of osteogenic differentiation, phase contrast images were captured using an inverted brightfield microscope (Leica Microsystem, Germany). Cells were then fixed with ice-cold methanol for 20 min, stained with 2% alizarin red solution in deionised water for 15 min and washed three times with deionised water. Brightfield images were acquired using an inverted microscope (Leica Microsystems, Germany). Semi-quantitative analysis of alizarin red staining was performed by dissolving the bound stain with 10% acetic acid. Samples were collected using a cell scraper and heated to 85°C for 10 min. Subsequently, 10% solution of ammonium hydroxide was used to adjust the pH to 4.5, and absorbance at 405 nm was read using a micro-plate reader (Varioskan Flash, ThermoFisher Scientific, Ireland).

To assess morphological changes of hBMSCs during cell expansion and during trilineage differentiation, cells were observed using an inverted brightfield microscope (Leica Microsystem, Germany). Phase contrast images were captured at different passages and during trilineage differentiation and were processed using ImageJ software (NIH, United States).