Carl axio imager m2

The viability of chondrocytes in GelMA and GelMA-GC hydrogels was determined on days 1 and 28 of free-swelling and day 14 of intermittent uniaxial loading (total 28 days of culture) using a live/dead assay described elsewhere.⁹⁷ (link) Briefly, after the push-out test, hydrogel constructs were sliced into two by a sterile scalpel, washed with PBS, and then incubated for 3 min at room temperature in PBS solution containing fluorescein diacetate (FDA; 1 μg/ml) and propidium iodide (PI; 1 μg/ml) (both from Sigma-Aldrich, USA). The samples were rewashed with PBS, and four Z-stack images per sample were acquired using a Zeiss Axio microscope (Carl Zeiss Axio Imager M2, GmbH, Germany) at each time point for each group (n = 4) and analyzed using Image J software (National Instruments, USA). Cell viability reported as a percentage of the total number of cells that were alive. Whole-slice images of hydrogels were also captured to represent an overall view of the impact of uniaxial loading on the top and bottom parts of hydrogel in cartilage-hydrogel constructs. To comprehensively examine the integration and cell viability of distinct cartilage-hydrogel areas, whole cartilage-hydrogel constructs were imaged on day 28 of culture with a Zeiss Axio microscope following a live/dead assay.

For examination of hyphal penetration and growth on leaves, leaf segments from a specified region (circular area with a radius of 1 to 4 mm from the primary inoculation site outwards) were collected after leaves were inoculated with mycelial homogenates. Lactophenol-trypan blue staining (10 mL lactic acid, 10 mL glycerol, 10 g phenol, 10 mg trypan blue, 10 mL sterile water) was used to visualize hyphae as previously described [28 (link)]. Samples were viewed and recorded using a light microscope (Carl Zeiss Axio Imager M2; Carl Zeiss Microscopy GmbH, Oberkochen, Germany).