Metaphase chromosomes were obtained from Mycetophylax conformis collected in Rio de Janeiro, and the Mycetophylax morschi cytotypes were defined according to the method proposed by Imai et al. [31 (link)]. The analyses were conducted on preparations obtained from 15 individuals per colony. The preparations were stained with Giemsa diluted in Sörensen buffer at (4%) for 20 minutes. On average, 10 metaphases were analyzed per slide. The best metaphases were photographed using an Olympus BX53 microscope equipped with a DP 73 Olympus® camera. The chromosome morphology was evaluated using the karyomorphometrical approach described by Cristiano et al. [20 (link)]. For this, we used the Image Pro Plus® software (Media Cybernetics, Rockville, MD) to measure each individual chromosome from the centromere to the end of the long (L) and short arms (S), as well as the total length (TL) of the chromosome. Chromosome length was averaged for the 10 individuals measured from each colony. The sum of the lengths of all the individual chromosomes constitutes the karyotype length (KL). The chromosomes were classified as metacentric (M), submetacentric (SM) or acrocentric (A) based on the arm ratio of Levan et al. [32 (link)] with the nomenclatural adjustments of Crozier [33 (link)].
Dp73 camera
Manufactured by Olympus
Sourced in Japan, United States
The DP73 is a digital microscope camera designed for high-performance imaging applications. It features a 12.8-megapixel CMOS sensor and supports a wide range of image capture and processing capabilities.
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Lab products found in correlation
Bx53 microscope, by Olympus (19 mentions)
Bx51 microscope, by Olympus (12 mentions)
Cellsens software, by Olympus (11 mentions)
Bx50 microscope, by Olympus (7 mentions)
Bx63 microscope, by Olympus (5 mentions)
Bx51 light microscope, by Olympus (5 mentions)
Bx43 microscope, by Olympus (5 mentions)
Bx41 microscope, by Olympus (4 mentions)
Bond polymer refine detection kit, by Leica (4 mentions)
Szx16, by Olympus (3 mentions)
Szx16 stereomicroscope, by Olympus (3 mentions)
Bx61 microscope, by Olympus (3 mentions)
Axio scope a1 microscope, by Zeiss (3 mentions)
Z1 axioscope microscope, by Zeiss (3 mentions)
Plan apochromat objective, by Zeiss (3 mentions)
Dako autostainer link 48, by Agilent Technologies (3 mentions)
Zen blue 2011, by Zeiss (3 mentions)
Ferangi blue chromogen kit, by Biocare Medical (2 mentions)
Mach4 universal hrp polymer kit, by Biocare Medical (2 mentions)
Anti cd68 mouse igg3, by Agilent Technologies (2 mentions)
142 protocols using dp73 camera
1
Karyomorphometric Analysis of Mycetophylax Ant Species
2
Immunohistochemical Analysis of Microglia Activation
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Histological diagnosis was revised and formalin-fixed paraffin-embedded representative sections for each patient were selected based on adequate tissue preservation as assayed by hematoxylin and eosin (H&E) staining and subjected to IHC. Briefly, 20μm thick paraffin embedded representative tissue sections were de-waxed, rehydrated and endogenous peroxidase activity blocked with 0,3% H2O2 in methanol for 20min. Antigen retrieval was performed by using a microwave-oven in 1mM Citrate buffer (pH 6.0). Sections were then washed in TBS (pH 7.4) and incubated for one hour or overnight in anti-Iba1 (rabbit polyclonal, 1:300; Wako), anti-CD68 (mouse IgG3, 1:100; Dako) or, anti-HLA-DR (1:250, Biomeda) in TBS 1% BSA. Signal was revealed using the DAKO Envision+System-HRP Labelled Polymer Anti-Rabbit or Anti-Mouse or the NovoLinkTM Polymer Detection System (NovocastraTM), followed by Diaminobenzydine (DAB) as chromogen and Hematoxylin as counterstain. For detection of microglia around the plaques, we combined silver staining with Iba1 IHC by using the MACH4 Universal HRP-Polymer kit (Biocare) and signal was revealed by Ferangi Blue Chromogen Kit (Biocare). Images were acquired with an Olympus Bx60 microscope and Cell Sens Standard Ink imaging software (Olympus Corporation) mounted on a DP73 Olympus camera.
3
Immunohistochemical Analysis of Microglia Activation
4
Ultraviolet Irradiation of Liquid Crystal Droplets
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Ultraviolet irradiation is achieved using a Hönle bluepoint LED lamp (λ=365 nm). MIX1 droplets with parallel surface anchoring are irradiated at intensity ≈50 mW cm−2, whereas MIX1 droplets with perpendicular surface anchoring are irradiated at intensity ≈180 mW cm−2. MIX2 droplets are irradiated with an intensity of ≈340 mW cm−2. Droplets are observed using Olympus BX51 polarized optical microscope and images are recorded by DP73 Olympus camera. The value of N is evaluated from crossed-polarized images before the ultraviolet light is turned on from the relationship N=d/δ where δ is the spatial period of the bulk onion-like structure at t=0 s, see Fig. 5a for the parallel case and Fig. 5j for the perpendicular case.
5
Quantitative Analysis of Muscle Fiber Morphology
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Photographs were acquired with a 10X objective using CellSens software, DP73 Olympus camera, and Nikon Eclipse E600 epifluoresence microscope. One entire cross-section from each muscle per sample was photographed and photomontages were manually assembled in Adobe Photoshop CC. Adobe Photoshop CC analysis tools were used to manually count all analyzable myofibers in the section according to positivity for MyHC 2b labeling and the presence of centrally-located nuclei. Cross-sectional area (CSA) was determined using the MATLAB application SMASH[29 (link)].
6
Immunofluorescence Analysis of DNA Repair Protein
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Control and IPF fibroblasts (n = 3, each) cultured on collagen in SF DMEM were irradiated at 9 Gy followed by incubation for an additional 6 h at 37 °C. Cells were then fixed with 4.0% paraformaldehyde in PBS and permeabilized in 0.1% Triton-X (MilliporeSigma) in PBS for 15 min. The cells were incubated with the anti-RAD51 antibody (catalog No. ABE257, MilliporeSigma) diluted 1:300 in PBS at 4 °C for 24 h. Cells were then incubated with an anti-rabbit Alexa Fluor 488 conjugated secondary antibody (catalog No. A11008, ThermoFisher Scientific) diluted 1:250 in PBS for 20 min. Cells on collagen matrix were cover slipped with ProLong Gold Antifade Mountant with DAPI (ThermoFisher Scientific). From each sample, 3 of ×20 magnified images were analyzed using an upright microscope (Olympus BX53 microscope, Olympus DP73 camera; Olympus America, Center Valley, PA, USA). Images analysis was conducted using ImageJ (NIH).
7
Immunofluorescence Analysis of Skeletal Muscle
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SM were carefully dissected, mounted on Tissue-Tek® O.C.T™ (Sakura Finetek, Hatfield, PA), and snap frozen in liquid nitrogen-cooled 2-methylbutane for 10–20 s. Samples were stored at −80 °C prior to cryosectioning. SM cryosections were washed with PBS and blocked with 0.05 % TBST (0.05 % Tween-20 in TBS) containing 10 % anti-goat serum. Slides were incubated with monoclonal anti-myosin (MYH7) (#M8421, 1:300; Sigma–Aldrich, St. Louis, MO) or anti-laminin antibodies (#PA1-16730, 1:500, Thermo Scientific, Waltham, MA) in blocking solution overnight at 4 °C. After washing with TBS, sections were incubated with secondary goat anti-rabbit Alexa Fluor-488 (#A-11008, 1:250) and anti-rabbit Alexa Fluor-594 (#A-11012, 1:250, both Thermo Fisher Scientific, Waltham, MA) antibodies in TBST plus anti-goat serum for 1 h at RT, followed by a 10-min incubation with DAPI. Slides were mounted with Dako Fluorescence Mounting Medium (Agilent Technologies, Santa Clara, CA) and visualized using an Olympus BX63 microscope equipped with an Olympus DP73 camera (Olympus, Shinjuku, Japan). The cross-sectional area (CSA) and Feret diameter of myofibers were determined using Fiji software (ImageJ® Version 1.52d; plugin “Muscle morphometry”). The areas of immunofluorescently stained fibers were quantified using ImageJ software (Version 1.53r).
8
Transwell Invasion and Migration Assay
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Invasion assays were performed using 24-well Transwell chambers (8.0-µm pore size; Corning, Inc.). Each Transwell chamber was coated with Matrigel matrix (Corning, Inc.) (100 µl at a dilution of 1:3 in DMEM; BD Biosciences), 24 h prior to use. Cells were cultured in DMEM for 24 h and then seeded onto cell inserts (2×104 cells/insert) in the upper chamber. Serum-free DMEM was added to the upper chamber, while DMEM (0.5 ml) containing 10% FBS was added to the lower chamber. After 24 h, the upper cells were removed, and the cells on the surface of the bottom chamber were fixed with methanol for 15 min and stained with 0.01% crystal violet (MedChemExpress) for 30 min at room temperature. Randomly selected areas were imaged with an inverted microscope (Olympus IX73; Olympus Corporation) connected to an Olympus DP73 camera (Olympus Corporation), and the number of cells was counted. The results represent the mean number of cells in five fields per membrane for triplicate inserts. Cell migration assays were conducted as the invasion assays, with the exception of the Matrigel coating.
9
Membrane Morphology Analysis of Bacterial Filtration
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The membrane morphology (structure, homogeneity/distribution) before and after filtration of bacterial suspensions was determined using scanning electron microscopy (SEM) imaging and optical microscopy. The SEM imaging of the samples’ surfaces and cross sections was performed using a FEI Sirion 400NC (FEI, Hillsboro, Oregon, USA) microscope at different magnifications. The membranes tested in the filtration experiments were immersed in 30 mL of 70% ethanol twice consecutively for half an hour in order inactivate viable bacterial cells and to fix them. Then, they were transferred to sterile Petri dishes and left to dry for 3 days at 37 ± 0.5 °C before further preparation for SEM analysis.
For light microscopy, selected non-tested membranes were cut into smaller square pieces (approx. 5 mm × 5 mm) and immersed in deionised water for 15 min at room temperature. Afterwards, the membrane pieces were dissected into individual slivers, which were transferred onto an objective glass, and observed under an Olympus SZ61TR stereomicroscope equipped with an Olympus DP73 camera (Olympus, Tokyo, Japan).
For light microscopy, selected non-tested membranes were cut into smaller square pieces (approx. 5 mm × 5 mm) and immersed in deionised water for 15 min at room temperature. Afterwards, the membrane pieces were dissected into individual slivers, which were transferred onto an objective glass, and observed under an Olympus SZ61TR stereomicroscope equipped with an Olympus DP73 camera (Olympus, Tokyo, Japan).
10
Immunohistochemical Localization of Amnion Epithelial Cells in Aorta
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Amnion epithelial cells were localised in the aorta using immunohistochemistry. Fixed (4% paraformaldehyde), paraffin-embedded thoracic aorta sections (5 μm) were dewaxed, incubated with histolene (2 × 10 min), rinsed with 100% and 70% ethanol and then distilled H2O. Antigen retrieval was performed using citrate buffer (pH 6.0), sections were then washed with PBS and endogenous peroxidase was blocked with 1% H2O2. Endogenous mouse IgG was blocked using goat anti-mouse IgG followed by blocking with 10% donkey serum in phosphate buffered saline. Sections were then incubated overnight at 4 °C with anti HLA-G (1:500; Ab52455; Abcam, UK). The next day, sections were washed and incubated with a horse radish peroxidase-conjugated donkey anti-mouse secondary antibody (1:200) for 45 min at room temperature. Sections were washed with PBS and incubated in DAB brown solution. Following this, sections were washed, counterstained with haemotoxylin and mounted with DPX mounting media. Images were captured with an Olympus DP73 Camera (Olympus Corporation, Tokyo, Japan) connected to an Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan) at 400 × magnification running CellSens Standard Software (version 1.17, Olympus Corporation).
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