Axiovision ac

Adult fish were anesthetized and placed on a 1% agarose plate with the caudal fins spread out. Zebrafish embryos, larvae and juveniles were anesthetized in E3 embryo medium containing 0.1 mg/ml tricaine. The plate was placed under a Leica MZ FLIII dissection microscope and images were taken using the AxioCam HSM digital camera and AxioVision AC software (Carl Zeiss). For live confocal imaging, fish were anesthetized and immersed in 0.17mg/ml tricaine in a petri dish. The caudal fins were flattened to the bottom of the petri dish with a slide hold-down (Warner Instruments 64–0248) and imaged with a water-immersion objective equipped on Nikon A1RsiMP Confocal. All images were processed using ImageJ (NIH).

Freehand cross sections of adventitious roots fixed in 70% EtOH were stained with 0.1% (w/v) berberine hemisulfate for 60 min, washed three times with distilled water and counterstained with 0.5% (w/v) aniline blue for a further 30 min for detection of CB (Brundrett et al., 1988 (link)). Stained sections were mounted in 0.1% (w/v) FeCl₃ in 50% (v/v) glycerine and examined using an Axioskop fluorescence microscope (Zeiss, Jena, Germany) with UV illumination and excitation filter G 365, chromatic beam splitter FT 395 and barrier filter LP 420. Pictures were taken with the AxioCam MRc (Zeiss) and picture recording software (AxioVision Ac, Version 4.4, Zeiss). Suberin exhibited a blue-white color under UV light. The development of CB in the anticlinal exodermal cell walls was determined and allocated to one of four stages: 0% (stage I), 0–25% (II), 25–50% (III) and 50–100% (IV) development of CB in the anticlinal cell wall of the exodermis.
Five roots without lateral roots were taken from each of the four replicates for cross-sectioning and 20 cells each from five cross sections were used for microscopic examination, therefore, the degree of development of CB was based on 400 cell walls per treatment.

After cryosectioning, muscle sections were stained with haematoxylin & eosin for assessment of muscle architecture, CD31 to visualize capillaries, Van Gieson’s stain to identify collagen and fibrosis, SDH activity as a general marker of mitochondrial (oxidative) activity, and periodic acid–Schiff stain for glycogen content^{75 (link)}. The muscle fibre cross-sectional area was measured after immunolabelling of laminin; fibre type was identified by staining myosin heavy chain I and myosin heavy chain IIa. Digital images of stained sections (four images per muscle section) were obtained using an upright microscope (20× objective) with a camera (Axio Imager D1, ZEISS) and AxioVision AC software (AxioVision AC, release 4.7.1, ZEISS) for acquisition. Myofibre cross-sectional area was quantified as described previously^{76 (link),77 (link)}.