Dfc420

Collagen content was analyzed using an optical microscope and image analyzer. Images were captured by a DFC 420 camera at 400 × magnification, which was attached to a Leica DM2500 trinocular optical microscope (Leica Microsystems, Wetzlar, Germany), and analyzed using Image-Pro Plus 4.5 software (NIH, Bethesda, MD, USA). To detect fibers, the optical density measurement method was used, and the software provided a threshold for positive areas and quantified them based on the determined area. Ten fields of alveolar septa per animal were examined. The results were expressed as a percentage of the positive area in comparison to the total area^{27 (link),28 (link)}.

The staining was performed according to the Standard Operating Procedure protocol at the Histopathology laboratory, Great Ormond Street Hospital, London. Sections were dewaxed in xylene, hydrated down through graded alcohol solutions to water, incubated for 10 min in 0.5% periodic acid, rinsed in distilled water, stained for 10 min with Schiff reagent, then rinsed in distilled water. Sections were then washed for 5 min in running tap water and counterstained in 1% eosin for 1 min, rinsed briefly in running tap water and dehydrated through ascending grades of alcohol. Sections were then cleared in xylene and mounted. For haematoxylin and eosin (H&E) staining, liver sections were processed according to standard protocols.
For quantitative PAS staining quantification, ten random images per liver section were captured with a microscope camera (DFC420; Leica Microsystems, Milton Keynes, UK) and software (Image Analysis; Leica Microsystems). Quantitative analysis was performed with thresholding analysis using the Image‐Pro Premier 9.1 software (Rockville, MD, USA).

Paraffin sections of stem bases were prepared as previously described (Xu et al., 2017 (link); Cheng et al., 2020 (link); Jin et al., 2022 (link)), with some modifications. The bases of ‘M9T337’ stem cuttings were collected at 6, 9, and 12 days after transplanting on 1/2 MS medium with 0.5 mg L-1 IBA and 0.1 mg L-1 NAA. The bases of tobacco stem cuttings were excised at 2, 4 and 6 days after subculture on hormone-free MS medium. The samples were fixed in FAA solution (70% ethanol: formaldehyde: acetic acid, 95:5:5 [v/v/v]) for 2 days at room temperature, and store at 4 °C. Then samples were dehydrated with a graded ethanal series (50%, 70%, 85%, 95%, and 100%), infiltrated with xylene, and embedded in paraffin. Cross sections with a 10 μm in thickness were cut with a Leica RM2245 (Leica Microsystems, Wetzlar, Germany) rotary microtome, transferred onto glass slides, deparaffined with xylene, and re-hydrated through an ethanol series, and stained with toluidine blue. Slides were observed using an optical microscope DM2500 (Leica Microsystems, Wetzlar, Germany) and photos were obtained using an attached digital camera DFC420 (Leica Microsystems, Wetzlar, Germany).