U hscbm

Samples of young capitulum at different developmental stages (0.5-4 mm) for histological sections were collected and fixed in FAA (Formalin–acetic acid–alcohol, 50% ethanol, 5% acetic acid, and 5% formaldehyde) at room temperature for 2 days, followed by embedding in paraffin after treatment with different concentrations of alcohol and xylene. The paraffin-embedded samples were cut in 8 µm (RM2265, Leica Biosystems, Wetzlar, Germany), then examined and photographed using the BX53 Microscope (OLYMPUS) equipped with a U-HSCBM (OLYMPUS) digital camera.

Skeletal muscle femoris were dissected, placed into 4% paraformaldehyde in phosphate buffered saline (0.01 M) for two days and then sequentially moved into increasing concentrations of ethanol (50, 70, 90 and 100%). Sections (7 µm) were mounted on glass slides and stained according to Mayer’s Hematoxylin - Eosin staining protocol. Digital images of the stained sections were used to identify the morphological features of the muscle. A bright-light microscope (Olympus U-HSCBM, Olympus, Tokyo, Japan) with a 10x magnification objective, digital camera and image capture software (cellSens Dimensions 1.11 software; Olympus, Tokyo, Japan) were used.

A time series of capitula of both the wild-type and gh cosmos were collected at early developmental stages for in situ expression analysis. Tissues were fixed by dehydration in an increasing gradient series of ethanol, clearing with xylene, and embedding in Paraffin [36 (link),37 (link)]. The sections were cut in 8 µm (RM2265, Leica Biosystems, Germany).
Gene specific probes were synthesized for CbLFY (307 bp) and CbUFO(300 bp) according to Braissant and Wahli, 1998 [38 ], using a PCR-amplified fragment with a T7 promoter sequence appended to the 5’ end and labeled following the instructions of the DIG RNA labelling Kit (Roche, Basel, Switzerland). Hybridization and detection were carried out as described by Neta et al. [39 (link)]. Sections were examined and photographed using the BX53 Microscope (OLYMPUS) equipped with a U-HSCBM (OLYMPUS) digital camera.