Using C. elegans as a Model in PKD
Jinghua Hu, Yong Yu in Polycystic Kidney Disease, 2019
ProtocolUse glow discharge formvar/carbon coated nickel grids (200 mesh, EMS cat no. FCF200-Ni). For negative staining; any type of formvar/carbon coated grids would work. Nickel grids are used here because gold or copper grids are not compatible with the following silver enhancement immunogold labeling protocol.Add 5 μL EV sample onto each grid, wait for 30 s, wick the solution with #1 Whitman filter paper from the edge of the grid, leaving a thin film of solution on the grid. Tilt the grid to get rid of remaining solution. However, do not touch the grid with filter paper, and do not let the grid dry before next step.Immediately add a drop Nano-W (Methylamine Tungstate solution nanoprobes cat no. 2018: http://www.nanoprobes.com/products/Negative-Stains.html) on the grid, wait for 1 min, wick the solution with Whitman filter paper, leave as little solution on grid as possible, but do not touch the grid with filter paper. Air dry and save the grid in a clean box for TEM.
Flow Cytometry
Wojciech Gorczyca in Atlas of Differential Diagnosis in Neoplastic Hematopathology, 2014
The results of the staining are determined by the comparison between negative controls and the intensity of staining with each antibody (Figures 3.14 through 3.16). The negative staining can be defined by the fluorescence intensity similar to that of negative controls (Figure 3.14). The staining is positive when the expression (fluorescence intensity) of any given marker (antibody) is greater than that of a negative (isotypic) control. In heterogeneous samples (e.g., BM specimen), the use of “built-in” negative controls can also be used, but sometimes this approach is limited, as different cell populations may display variable intensity of “background” (nonspecific) staining. Some cells (e.g., monocytic cells, atypical promyelocytes, and large lymphomatous cells with decreased viability) often display high nonspecific staining (Figures 3.14 and 3.15). Therefore, the threshold between positive and negative expressions should be established for each cell population based on the control (negative) sample and not by comparison with other population known to be negative for a specific marker. As illustrated in Figure 3.14, if only staining with CD14 was performed, each population with intensity greater than that observed in lymphocytes (red dots) which do not express CD14 would be considered positive. However, as it is evident from the staining with negative (isotypic) control antibody (Figure 3.14A), the abnormal cells (green and blue dots) have very high nonspecific staining that is similar to that observed with CD14 (Figure 3.14B). Figure 3.15 illustrates the different level of background (nonspecific) staining among different populations (myeloblasts, granulocytes, and benign T lymphocytes) in the sample from the BM involved by AML. Only careful comparison of antigen expression for each population with that of nonspecific staining (control) allows for the characterization of identified cells.
Carbohydrate Histochemistry
Joan Gil in Models of Lung Disease, 2020
A negative staining result at the ultrastructural level is particularly difficult to interpret. As a rule, preliminary studies at the light microscopic level should precede electron microscopic cytochemistry to establish the presence in the cell of the entity under study. Only the very exceptional cell or tissue constituent cannot be visualized histochemically by careful observation under the light microscope.
Maximal number of pre-synaptic ribbons are formed in cochlear region corresponding to middle frequency in mice
Published in Acta Oto-Laryngologica, 2018
Le Yang, DaiShi Chen, TengFei Qu, TongHui Ding, AiHui Yan, Pinggui Gong, Yunyi Liu, Junjun Zhang, ShuSheng Gong, ShiMing Yang, Hong Peng, Ke Liu
The samples were fixed in 4% paraformaldehyde and dissolved in 0.1 M PBS with 30% sucrose (pH 7.4) for 1 h at room temperature. They were washed three times in 0.01 M PBS and pre-incubated for 30 min at room temperature in blocking solution of 5% normal goat serum in 0.01 M PBS with 0.3% Triton X-100. Next, the samples were incubated with a combination of anti-CtbP2 (1:50, Santa Cruz) and left at 4 °C overnight. After incubation, the samples were washed in 0.01 M PBS three times, and incubated with the secondary antibody IgG FITC identifying anti-CtBP2 (1:100, Santa Cruz) at 37 °C for 40 min. After incubation, the samples were washed in PBS twice. Approximately 40 μl of DAPI (4′,6-diamidino-2-phenylindole; Santa Cruz) was applied to the slide. The basement membranes were mounted onto a dissecting microscope. The samples were imaged directly with fluorescent microscopy to test the specificity of the primary antibody. Controls were performed to demonstrate negative staining (data not shown).
Identification of tumor-infiltrating immune cells and prognostic validation of tumor-infiltrating mast cells in adrenocortical carcinoma: results from bioinformatics and real-world data
Published in OncoImmunology, 2020
Xi Tian, Wenhao Xu, Yuchen Wang, Aihetaimujiang Anwaier, Hongkai Wang, Fangning Wan, Yu Zhu, Dalong Cao, Guohai Shi, Yiping Zhu, Yuanyuan Qu, Hailiang Zhang, Dingwei Ye
To further validate TIMCs’ prognostic significance, real-world data were also collected from our institute. This study included 39 ACC patients who underwent surgical treatment from Fudan University Shanghai Cancer Center (FUSCC) between 2013 and 2019, and tumor specimens were obtained with informed consent. Anti-tryptase monoclonal antibody (Ab2378, diluted 1:10,000; Abcam) was used to identify mast cells using immunohistochemistry (IHC). The positive or negative staining was evaluated by two experienced pathologists and determined as follows. The overall IHC score from 0 to 12 was evaluated according to the multiplying of the staining intensity and extent score, as previously described.20 The IHC scores 0–3, 4–12 are defined as low TIMC group and high TIMC group. Scatter diagram was drawn to explore the correlations between TIMC abundance with phenotype, and Kaplan-Meier method was applied to validate TIMC’s prognostic significance by comparing two groups survival rates.
Washingtonia filifera seed extracts inhibit the islet amyloid polypeptide fibrils formations and α-amylase and α-glucosidase activity
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Sonia Floris, Antonella Fais, Rosaria Medda, Francesca Pintus, Alessandra Piras, Amit Kumar, Piotr Marek Kuś, Gunilla Torstensdotter Westermark, Benedetta Era
In addition, negative staining was made for analysis by Transmission Electron Microscopy (TEM), a useful technique for assessing the morphology of in vitro formed amyloid fibrils from proteins or peptides that allows researchers to see structural features at the nanometre scale that cannot be visualised by light microscopy. Negative staining typically generates the sample with good contrast and well-preserved morphology. The stain forms a coating over the sample that appears light and the surrounding stain appears dark. Figure 4 showed that at 1:5 IAPP to extract ratio (MES 200 µg/mL), the formation of IAPP fibrils is inhibited.
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