Axiovert 135

For visualising dead cells within MCTS, spheroids were stained for 30 min under cell culture conditions with 6 µg/mL propidium iodide (PI) which stains dead cells only because of their permeabilised cell membranes [12 (link)]. MCTS were then rinsed with PBS to remove excess PI. Immediately thereafter, brightfield and fluorescence images were acquired by inverted fluorescence microscopy using a Zeiss Axiovert 135 fluorescence microscope and AxioVision software. ImageJ was used for further image processing.

Sex chromosomes of H. punctata were selected to be used as probes since this species exhibits the closest 2n relative to the proposed ancestral state for the genus and, at the same time, it possesses a multiple sex chromosome system of the X₁X₁X₂X₂/X₁X₂Y type^{28 (link),32 (link),34 (link),35 (link),42 (link)}. Fifteen copies of the X₁ and X₂ chromosomes were isolated by glass-needle-based microdissection under an inverted microscope (Zeiss Axiovert 135). The collected DNA material was then amplified in a primary degenerated oligonucleotide-primed polymerase chain reaction (DOP-PCR)⁷⁶ . The probes were then labeled in the secondary DOP-PCR reaction using 1 µL of the initial amplified product as a template DNA⁷⁷ . The probe derived from the X₁ chromosome (HPU-X₁) was labeled with Spectrum Orange-dUTP (red), and the one derived from the X₂ chromosome (HPU-X₂) with Spectrum Green-dUTP (green) (Vysis, Downers Grove, United States).
The 5S and 18S rDNA fragments were obtained by PCR from the wolf fish Hoplias malabaricus genome using primers and thermal profiles described in previous studies^{78 (link)–80} . The labelling was done by nick translation using Atto550-dUTP (red) for the 5S rDNA and Alexa Fluor 488-dUTP (green) for the 18S rDNA (both Jena Biosciences, Jena, Germany), according to manufacturer's protocol.

Trypan Blue exclusion assay was used for counting of living cells and monitoring of cell proliferation. CAD cells were seeded in 35-mm dishes (2 × 10⁴ cells/dish) and incubated for 24, 48, or 72 h in the absence or presence of 4 mM or 8 mM Phe. For the assay, Trypan Blue solution was added to cell culture (final concentration 0.75%) at room temperature for 3 min, cells were transferred to a Neubauer Chamber, and living and dead cells were counted using an optical inverted microscope (model Axiovert 135, Carl Zeiss; Jena, Germany).

Based on the cell viability assay, the apoptosis-inducing effect of ADP extract was evaluated at two effective doses viz. 15 and 25 mg/mL. DNA condensation was measured using Hoechst 33258 staining as per a previously published method^{21 (link)}. To assess nuclear morphology, stained cells were captured under an inverted fluorescence phase contrast microscope (Zeiss AxioVert 135, USA).

Fixed samples were sub-sampled three times and were diluted 1:10 in tap water. One ml was transferred to an Uthermol's chamber for microscopic analysis. An inverted microscope (Zeiss Axiovert 135) was used to identify and count (Zeiss EC Plan-Neofluar 40x/0.75 objective, Zeiss EC Plan-Neofluar 100 × 1.3 Oil objective, if necessary) phototrophic organisms at the level of genera and, if possible, species within three fields of vision. For samples obtained from laboratory experiments, semi-quantitative abundance was described as dominant (‘5', >30 of the cell number counted), frequent (‘4', 10–30%), regular (‘3', 3–10%), scarce (‘2', 1–3%) and sporadic (‘1', <1%) based on two taxonomic references and the recommendations by the Swiss Federal Office for the Environment33 34 35 . For field samples, presence-absence data were documented.

Lungs were fixed in 4% paraformaldehyde (PFA) in 0.1 M Na₂HPO₄ and 23 mM NaHPO₄ (pH 7.4) for 4 h on ice. The tissue was then immersed in ice-cold 20% sucrose in 0.1 M Na₂HPO₄ and 23 mM NaHPO₄ (pH 7.4) overnight to cryoprotect the tissue, mounted using OCT, and transversal 4–6 μm sections were obtained with a cryostat. Tissue sections were stained with immunofluorescence to assess pulmonary vascular remodeling and Masson trichrome stain to assess pulmonary fibrosis. The images were acquired using light microscopes (Axiovert 135, Zeiss, and Nikon Eclipse E 400) or with a laser scanning confocal microscope (Olympus). Pulmonary fibrosis was quantified using a grid that divided the field of view into 100 squares, the number of collagenous tissue (blue stain) in the grid was scored as 1 (present) or 0 (absent). Results are expressed as the percentage occupied by fibrosis to the total area examined.