Infinity2 camera

Gram staining was performed on stool samples using strains of Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) as standards. Catch-All sample collection swabs (EPICENTRE Biotechnologies, Madison, WI) were used to smear samples on glass slides, and stool slides were swabbed with phosphate-buffered saline (PBS) to reduce the bacterial concentration. The protocol of the Gram staining kit manufacturer (Remel, Lenexa, KS) was followed for staining, with no changes. Slides were viewed under an Olympus BX-41 microscope using the 100× oil immersion objective. Images were captured using a Lumenera INFINITY2 camera.

For experiments analyzing locomotor capacity, worms were synchronized by allowing gravid adults to lay eggs in a 2-h window, and eggs were allowed to grow to 3 days or 7 days, depending on the experiment (Figure 1B). For experiments of neuromuscular function, aged wild type and mutant hermaphrodites were examined for their sensitivity to an acetylcholine esterase inhibitor, aldicarb (Sigma). Three plates of 20 age-matched animals per genotype were transferred onto NGM plates containing 1 mM aldicarb. Movement was scored every 15 min for 2 h, and genotypes were blind to the scorer. For analyses of 7-day-old animals, worms were transferred to FUdR-containing plates at 3 days to prevent new progeny from developing.
Thrashing and body bend assays were performed to test for locomotor capacity. On test days, animal behavior was recorded by video using an Infinity 2 camera (Lumenera). For thrashing assays, individual worms were added to M9 solution, allowed to equilibrate for 1 min, and scored the next 15 s for the number of thrashes (this number multiplied by four to get thrashes per minute). For body bending assays, individual worms were transferred onto a NGM plate containing E. coli (HB101), and scored the next 30 s for the number of bends (this number was doubled to get body bends per minute).

Dissected bones from mice aged ≥2 wk were formalin fixed for 48 h at RT, or 8-wk-old bones were fixed for 72 h at 4°C before decalcification in 14% EDTA (Sigma-Aldrich). Embryonic and newborn bones were fixed in 4% paraformaldehyde for 24 h at 4°C. All tissues were embedded in paraffin, sectioned at 4 µm, placed on Superfrost slides, and stained with hematoxylin and eosin (H&E). Unstained sections were deparaffinized and hydrated with water for further staining. Safranin O staining was performed as described previously (Mahmoodi et al., 2005 (link)) with a few modifications. Briefly, slides were stained with Weigert’s iron hematoxylin (BDH), 0.001% fast green solution (BDH), 1% acetic acid, and 0.1% Safranin O (Thermo Fisher Scientific). Stained specimens were photographed using an Infinity2 camera (Lumenera) and Olympus SZ2-1LST dissecting microscope (uEye software; 4×/0.5-NA objective) or Zeiss Axiolab microscope (Zen blue software; 20×/0.5-NA or 2.5×/0.075-NA objective) at RT.

Tube formation assays were performed as previously described (54 (link)). Briefly, Matrigel (BD Biosciences) was diluted 1:3 in medium, and 300 μL was added to each well of a 12-well plate and incubated at 37°C for 30 minutes to allow polymerization. Cells were suspended in the same medium at a density of 5 × 10⁴ cells/well, and 400 μL of the cell suspension was added to each well. Photographs were obtained after 6 hours using a Nikon Eclipse TE2000-S microscope with a Nikon Plan Fluor 4× objective lens. The images were captured using an INFINITY2 camera (Lumenera) and analyzed by ImageJ (NIH).