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Histochemical Study of Estrogen Receptors in the Rat Uterus With a Hydrophilic Fluorescent Estradiol Conjugate
Published in Louis P. Pertschuk, Sin Hang Lee, Localization of Putative Steroid Receptors, 2019
The principle of the fluorescent steroid histochemical techniques is based on the assumption that steroid hormone molecules are capable of binding to their receptors, soluble or insoluble, even when the hormone is covalently coupled to a dye or a carrier as long as its physiologically active determinants or radicals are exposed. Such a possibility was initially demonstrated with fluoresceinated 6-carboxymethyl oxime estradiol-170.40 However, because of its low solubility and hydrophobic nature that often cause poor differential staining in tissue sections, this compound has not been widely tested.
The Noncollagenous Proteins of the Intervertebral Disc *
Published in Peter Ghosh, The Biology of the Intervertebral Disc, 2019
Elastic fibers are composed of two distinct morphologic components.18,87–90 a central amorphic and a microfibrillar outer region. These two components may be distinguished on the basis of their differential staining characteristics with uranyl acetate and lead salts, which stain the microfibrillar structures, but not the dense amorphous central component.18 The amorphous and microfibrillar components are chemically unrelated, which is apparent by their differential staining characteristics. The former is rich in hydrophobic amino acids such as glycine, alanine, proline, and valine, while the microfibrillar component is much more acidic and apparently has a high content of cysteine and a high glycoprotein content.23,87,88 Collagen fibrils which occur associated with elastin fibrils in AF tissue may be distinguished from microfibrillar components by their specific staining with uranyl acetate or phospho-tungstic acid and the lack of staining with lead salts.
The Identification of Cell Types in the Normal Adult Colon
Published in Leonard H. Augenlicht, Cell and Molecular Biology of Colon Cancer, 2019
The structure of goblet cells directly reflects their secretory function. The cells are highly polarized and compartmentalized, with regular arrays of rough endoplasmic reticulum (RER) peripheral and subjacent to the nucleus within the basal cytoplasm, a well developed supranuclear Golgi complex, and an apical accumulation of mucin packed secretory granules (Figures 4 to 6). In most regions of the gastrointestinal tract, the goblet cell nuclei and cytoplasm stain intensely basophilic at the light level (Figure 4) and electron dense ultra-structurally (Figure 6). We have observed, however, that structurally comparable cells in the human proximal (Figure 5) and distal colon frequently have a pale nucleus and cytoplasm. The functional basis for this differential staining and electron density is unknown.
Antiproliferative Effect of Trifolium Pratens L. Extract in Human Breast Cancer Cells
Published in Nutrition and Cancer, 2019
Mozafar Khazaei, Mona Pazhouhi
Apoptosis was evaluated by detection of DNA fragmentation using an In Situ Cell Death Detection Kit, AP (Roche Diagnostics; Germany) and according to the manufacturer’s instructions. Briefly, after 48 h treatment with T. pratense extract in a 96 well plate, the cells were fixed using 4% paraformaldehyde in PBS (freshly prepared) for 1 h, permeabilized using a solution (0.1% Triton X-100, 0.1% sodium citrate) for 5 min on ice, and incubated with 50 μl of TUNEL mixture solution (label and enzyme solution) at 37 °C for 1 h in a humidified incubator. For differential staining of the cells the PI staining solution was added and the plate was incubated for 4 min at room temperature. Finally, cells were analyzed using a fluorescence microscope. All the mentioned stages are performed in dark condition. The apoptotic index of the cells was calculated as follow:
Sterigmatocystin-induced DNA damage triggers cell-cycle arrest via MAPK in human neuroblastoma cells
Published in Toxicology Mechanisms and Methods, 2021
Veronica Zingales, Mónica Fernández-Franzón, Maria-José Ruiz
Micronucleus (MN) assay was carried out using the Litron In Vitro Microflow Kit (Litron Laboratories, Rochester, NY), according to the OECD TG 487. Conditions set for this assay point that it must be carried out exposing logarithmically dividing cells to the toxic for a duration of time that approximates 1.5–2 normal cell cycles. At this time, cells must be harvested and processed for MN scoring. The assay was performed according to manufacturer’s instructions based on previous reports (Bryce et al. 2007, 2008). Briefly, 7 × 105 cells/well were seeded in six-well plates and exposed to 0.78, 1.56, and 3.12 µM of STE for 48 h, which is equal to 1.5–2 doubling times for this cell line. The day of the experiment, cells were stained with Nucleic Acid Dye A Solution and placed on ice near a light source for 30 min, to induce the photoactivation of the EMA flurorochrome dye, a reagent that crosses the compromised outer membrane of apoptotic and necrotic cells. Afterwards, cells were washed, lysed with the Litron Lysis kit-solution and preserved from light for 60 min. During the lysis step, cytoplasmic membranes were digested to liberate nuclei and MN. Finally, a lysis solution 2 containing Nucleic Acid Dye B (SYTOX fluorochrome), which labels all chromatin, was added and cells were incubated for 30 min at room temperature in the dark. The differential staining allows to distinguish between healthy chromatin and dead/dying cells. Gating and analysis strategies were performed by using a BD FACSVerse (BD Biosciences, Franklin Lakes, NJ) flow cytometry, following the instructions and templates provided by the Litron In Vitro Microflow Kit manual and as described by Bryce et al. (2010, 2008). Three independent experiments were performed.
VBNC, previously unrecognized in the life cycle of Porphyromonas gingivalis?
Published in Journal of Oral Microbiology, 2022
A Progulske-Fox, SS Chukkapalli, H Getachew, WA Dunn, JD Oliver
Detection of VBNC. An array of viability markers have been used to differentiate viable and VBNC cells. The most common method is differential staining combined with direct microscopic enumeration, the LIVE/DEAD® Bac Light™ assay. This assay employs two fluorescent dyes, Syto 9 and propidium iodide, which have variable cell permeability characteristics that can differentiate cells with different membrane integrities [23].