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Carbohydrate Histochemistry
Published in Joan Gil, Models of Lung Disease, 2020
Bradley A. Schulte, Russell A. Harley, Samuel S. Spicer
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.
Using C. elegans as a Model in PKD
Published in Jinghua Hu, Yong Yu, 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.
Microbiological Diagnosis of Viral Diseases
Published in Nancy Khardori, Bench to Bedside, 2018
The small size of viruses makes direct visualization under a light microscope of limited value in a diagnostic virology laboratory. Viruses can be visualized by negative staining under the electron microscope, but this facility is available only in research and limited number of tertiary care centres.
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.
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).
Polymeric micelle nanocarriers for targeted epidermal delivery of the hedgehog pathway inhibitor vismodegib: formulation development and cutaneous biodistribution in human skin
Published in Expert Opinion on Drug Delivery, 2019
Somnath G. Kandekar, Mayank Singhal, Kiran B. Sonaje, Yogeshvar N. Kalia
Morphology of the drug-loaded micelle nanocarriers was observed by transmission electron microscopy (TEM) using a FEI TecnaiTM G2 Sphera (Eindhoven, The Netherlands) equipped with a 2000 × 2000 pixel high-resolution digital camera. The sample preparation was performed using the negative staining method. Briefly, 5 μL of the micelle solution was placed onto an ionized carbon-coated copper grid (0.3 Torr, 400 V for 20 s), subsequently the grid was placed in contact with a 100 μL drop of a saturated uranyl acetate aqueous solution for 1 s and then again in a second 100 μL drop for 30 s. The excess staining solution was removed and the grid was dried at room temperature prior to visualization by TEM.