Axiovision imaging system software

Stomatal density and aperture were evaluated over the course of the trial by epidermal imprints following weekly gas exchange measurements. Once a gas exchange measurement was complete, a small drop of transparent acrylic lacquer was lightly spread onto abaxial leaf surfaces at the leaf tip and mid-leaf section where gas exchange measurements were performed. After the lacquer had dried to the touch, a piece of clear tape was used to remove the imprinted acrylic layer and attach it to a microscope slide. Photos of the leaf surface imprints were taken with a Zeiss SteREO Lumar V.12 fluorescence stereomicroscope (Carl Zeiss Microimaging GmbH, Jena, Germany) with Zeiss AxioCam camera, model MRm 1.4 MP CCD. Images were preprocessed using the AxioVision Imaging System software (Carl Zeiss Microimaging GmbH, Jena, Germany, version 4.9), adjusting the contrast to improve definition of guard cells and stomatal aperture. Images were captured at 200× magnification. Using the NIH open-source image analysis software Image J (Version 1.53a), stomatal density and aperture size were accessed at two locations (tip and mid-leaf) on abaxial surfaces at the five time points during the trial (T0, T1, T2, T3 and T4) when gas exchange measurements were performed.

Cells and tissue were fixed in 2% PFA and processed for histology and immunocytochemistry as previously described (Gargioli et al., 2008 (link)). Briefly, MP were cultured in fibronectin-coated 8-well chamber slides (Nunc) as described above. After fixation with 2% PFA for 10 min, cells were incubated overnight at 4°C with the following primary antibodies: anti-α-smooth muscle actin (SMA; Dako), platelet-derived growth factor receptor beta (PDGFR-β; Cell Signaling Technology), chondroitin sulfate proteoglycan (NG2; Merk Millipore) and myosin heavy chain (MyHC; DHSB), diluted in accordance with the manufacturers' instructions. Cells were then incubated with the appropriate secondary fluorophore-conjugated antibody. Nuclei were stained with DAPI. Fluorescence photomicrographs were taken with an Axio Observer A1 microscope equipped with a fluorescence detection system at 20 × magnification. Imaging analysis was performed with AxioVision Imaging System software (Zeiss).