Dc500 digital camera

After sacrificing the animals, the eyes were enucleated, and the anterior eye segments dissected and quenched in light petroleum chilled with an acetone-dry ice mixture. Sections were cut on a cryostat and transferred to glass slides. Subsequently, the cryostat sections were fixed in acetone at 4°C for 5 minutes. For the detection of MDA, NT, and iNOS, the postfixed cryostat sections (in acetone at 4°C for 5 minutes) were incubated (60 minutes) with monoclonal mouse anti-iNOS (Biosciences, San Jose, CA, USA) or monoclonal mouse anti-NT (Abcam, Cambridge, UK) or polyclonal goat anti-MDA (US Biological, Swampscott, MA, USA), followed by 45 min incubation by Alexa Fluor 488 Goat Anti-Mouse IgG or Alexa Fluor 488 Donkey Anti-Goat IgG (Thermo Fisher Scientific, Waltham, MA, USA). Following the procedure, the samples were immediately examined using the microscope Leitz (Orthoplan Leitz light microscope equipped with a Leica DC 500 digital camera).

Intracellular NO content in Col-0 TCLs cultured with either 10 μM IBA or 10 μM IAA was quantified using the cell-permeable diacetate derivative diamino-fluorescein-FM (DAF-FMDA; Sigma) under epifluorescence microscopy. TCLs were incubated in 20 mM HEPES/NaOH buffer (pH 7.4) supplemented with 5 μM DAF-FMDA for 20 min [26 (link)] at 2, 3 and 6 days of culture, after having verified that no significant epifluorescence signal was detectable with the buffer alone (Additional file 1: Figure S1 a-b). After washing three times with the buffer to remove the excess of the fluorescent probe, TCLs were observed using a Leica DMRB microscope equipped with the specific set of filters (EX 450–490, DM 510, LP 515). The images were acquired with a LEICA DC500 digital camera and analysed with the IM1000 image-analysis software (Leica). Ten observations in each of 20 TCLs per treatment were randomly carried out, and the intensities of the fluorescence signal (in green colour) were quantified using the ImageJ software (National Institute of Health, Bellevue, WA, USA) and expressed in Arbitrary Units (AUs; from 0 to 255). The values were averaged and normalized to the control ones, i.e., to those measured in TCLs incubated in the buffer without the fluorescent probe.

Loaded tibiae (n = 4) were dissected, fixed in 4% formaldehyde (Alfa Aesar Inc., Ward Hill, MA, USA), and stored in 70% EtOH. Before embedding, tibiae were washed, dehydrated through graded concentrations of alcohol, infiltrated, methyl methacrylate embedded, and sectioned at a thickness of 7 μm. Fluorochrome labels were observed using a Leica Q550IW fluorescence microscope with a DC 500 Leica digital camera. The distance between the inner and outer fluorescent labels at increments of approximately 5-μm part was measured in multiple sections per mouse and averaged. Comparable images from microCT-based morphometry and histomorphometry were visualized. MAR was calculated by dividing this distance by the labeling period (7 days). In addition, the MAR calculated from 3D microCT-based morphometry was quantified by measuring the average and maximum thickness of formed surfaces and plotted against histomorphometrically based MAR, and their differences were visualized using the Bland-Altman plot.