Image pro plus program

The FISH experiments were carried out on specimens ZUEC 17569 and ZUEC 17579
(Table S2),
which represent the populations of Rio de Janeiro and Duque de Caxias,
respectively. The PcP190 satellite DNA sequence previously isolated from
C. gaudichaudii by Vittorazzi et al. (2014 (link)) was amplified to obtain chromosomal probes. For this,
one cloned fragment was amplified by PCR in the presence of digoxigenin-dUTP
(Roche) and primers T7 and SP6, which flank the connection site of the pGEM-T
Easy Vector (Promega). The probes were mixed with salmon DNA (1 ng/μL of probe)
and precipitated in ethanol. The DNA was dissolved in a hybridization buffer at
pH 7 composed of deionized formamide (50%), 2 x SSC, phosphate buffer (40 mM),
Denhardt’s solution, SDS (1%), and dextran sulfate (10%). The in
situ hybridization technique was based on Viegas-Péquignot (1992 ), with modifications for the detection
of digoxigenin-labeled probes with anti-DIG-Rhodamine (Roche).
The microsatellites (CA)₁₅ and (GATA)₈ oligonucleotides
were marked directly with Cy5-fluorochrome at the 5’ end during synthesis
(Sigma-Aldrich) and used as probes in FISH assays that followed the protocol of
Kubat et al. (2008 (link)), under high
stringency (77%) conditions. Images of the hybridized metaphase chromosomes were
captured with an Olympus BX-60 microscope and edited with the Image-Pro Plus
program (Media Cybernetics).

The right postcaval lobes were fixed in formalin and embedded in paraffin. Then, the embedded lung lobes were sectioned in thickness of 5 μm, stained with haematoxylin and eosin (H & E) and photographed using a Olympus CKX41 microscope (Olympus, Japan) fitted with an Olympus DP20 camera connected to a computer. The Image Pro Plus program (Media Cybernetics, USA) was utilized to objectively assess the level of inflammation present in each image. The inflamed areas stained a more intense purple than the noninflamed areas. The mean percent of area inflamed was quantified averaging from three to five lung sections of each of the different groups of mice.

Confluent cultures of Trichomonas vaginalis plated on 35 mm Petri dishes were injected with Lucifer Yellow CH (5% in 150 mM LiCl) (457.2 Da) using glass microelectrodes (resistance between 40 and 70 MΩ) by short hyperpolarizing current pulses (0.1 nA, 100 milliseconds using a WPI amplifier, model 7060; USA). Fluorescence was observed on an Axiovert 100 microscope (Carl Zeiss, Oberkochen, Germany) equipped with appropriate filters (Zeiss BP450-490/FT510/LP520), and micrographs were taken using the Image Pro Plus program (Media Cybernetics, Rockville, MD, USA) 2 min after dye injection [20 (link)]. A minimum of 120 cells were injected in at least four independent experiments to determine the degree of coupling.

The effect of treatments on human skin cell migration was determined using a standard scratch-wound assay. Cells were seeded at 2 × 10⁵ cells/well in a 12-well plate and incubated at 37 °C overnight. Once the cells were confluent, wounds were created on the cell monolayers using an Incucyte WoundMaker (Sartorius, Germany). Then, the cells were washed with PBS before adding treatments and DMEM with 10% FCS. Cells were incubated and imaged every 3 h using the Olympus IX83 Fluorescence Microscope (Olympus, Tokyo, Japan) for 12 h and again at 24 h and 30 h. The distance between cell fronts was measured using the ImageProPlus program (Media Cybernetics Inc., Bethesda, MD, USA) and percentage wound closure was calculated.

Myocardial fragments were fixed in 4% buffered formalin, processed by a standard technique, and paraffin-embedded sections (3–4 μm thick) were stained with hematoxylin and eosin, Masson’s trichrome (Bio-Optica), Sirius Red (Bio-Optica) and used for histological examination. Masson’s trichrome-stained sections were used in total for morphometric evaluation of percent fibrosis at × 100 using the Image-Pro Plus program (version 6.0.0.260; Media Cybernetics, USA). Sirius Red-stained sections were used for a semi-quantitative 5-point assessment of the amyloid deposits distribution in the interstitium around blood vessels and cardiomyocytes: 0—no deposits; 1—deposits were around single cardiomyocytes; 2—around small groups of cardiomyocytes; 3—around half of cardiomyocytes and single vessels; 4—around more than 75% of cardiomyocytes and blood vessels. The slides were examined by light microscope Leica DMRB with Leica DFC495 camera and Leica PL FLUOTAR 10 × /0.3 and Leica N PLAN 40 × /0.65 (Leica Microsystems Gmbh, Austria).