Diagnostic applications of immunology
Gabriel Virella in Medical Immunology, 2019
The principles of quantitative immunofluorescence are similar to those described for indirect fluorescence assays: antigen is bound to a solid phase, exposed to a serum sample containing specific antibody, unbound immunoglobulins are rinsed off, and a fluorescein-labeled antibody is added to reveal specific antibody. A fluorometer is used to measure the amount of fluorescence emitted by the second antibody. Since the amount of fluorescent antibody added to the system is fixed, the amount that remains bound is directly proportional to the concentration of antibody present in the sample. Thus, a quantitative correlation can be drawn between the intensity of fluorescence and the concentration of antibody. These quantitative tests have been adapted to microbiological assays and can identify IgG and IgM antibodies.
Pathology and Epidemiology
John T. Kemshead in Pediatric Tumors: Immunological and Molecular Markers, 2020
In the earlier days of the application of immunohistological methods to tumor diagnosis, the method used to demonstrate the site of antigen localization was immunofluorescence using the action of ultraviolet light on antibodies conjugated with fluorescein or other fluorogen. Indeed, immunofluorescence is still the method of choice in some areas of study because of its high degree of sensitivity and ability to locate small quantities of antigen. It does suffer from disadvantages in the short storage life of the sections and the inconvenience of requiring a separate facility for ultraviolet light microscopy in addition to the white light microscopy available in most laboratories.
Renal Disease; Fluid and Electrolyte Disorders
John S. Axford, Chris A. O'Callaghan in Medicine for Finals and Beyond, 2023
Damage to the glomerular filtration barrier causes a protein leak and the nephrotic syndrome. Light microscopy and immunofluorescence are normal. Electron microscopy shows glomerular epithelial podocyte foot process fusion (Figure 8.11). The cause is unknown, but it can occur with SLE or use of NSAIDs.
Therapeutic effects of thymoquinone or capsaicin on acrylamide-induced reproductive toxicity in rats mediated by their effect on oxidative stress, inflammation, and tight junction integrity
Published in Drug and Chemical Toxicology, 2022
Ekram Nemr Abd Al Haleem, Walaa Yousef Soliman Hasan, Hossam Mohamed Mohamed Arafa
Immunofluorescence is a technique for detecting antigens and antibodies in which the antibodies are labeled with fluorescent dyes, and the resulting antigen-antibody complex is visualized using a fluorescent microscope. The indirect immunofluorescence assay was used in this research (Odell and Cook 2013). Occludin Antibody (E-5) was obtained from Santa Cruz Biotechnology, which is a mouse monoclonal IgG2b (kappa light chain) (Oregon, USA). Using a Leica fluorescence microscope (Model: Leica DM 5500B, Leica Microsystems, Wetzlar, Germany), tissue sections were examined and imaged in blue, green, and red channels. The fluorometric analysis of the results was performed using Image-J software on a minimum of five fields from each rat testicular segment (minimum of two rats from each group) (NIH, USA).
Salivary myoepithelial cells: an addendum
Published in Ultrastructural Pathology, 2018
Asterios Triantafyllou, Lauge Hjorth Mikkelsen, Douglas R. Gnepp, Simon Andreasen, Jennifer L. Hunt, Kenneth O. Devaney, Vincent Vander Poorten, Alessandra Rinaldo, Stefan M. Willems, Alfio Ferlito
Electron microscopy has made valuable contributions to the investigation of the particular appendages and processes associated with the salivary myoepithelial cells, examined in the present article. It is unlikely that the discovery of cilia and microcalcification in these cells would have been possible without electron microscopy. However, the relevant investigations have seemingly reached a plateau. Although the processes examined are of academic interest, they do not seem of primary clinical importance, which places them on the fringe of current biomedical research. Furthermore, immunofluorescence with different dyes to label various cellular components in conjunction with computer-aided technologies and light microscopical, double immunohistochemistry, being cost-effective and less time intensive, are currently given preference as investigative modalities. Double immunoelectron microscopy together with technological advances enabling automation of processing and sectioning of tissues for electron microscopy may herald a revival of electron microscopy. It is only hoped that the present article may complement previous reviews1,2,4,5 and so stimulate further research.
Extracellular vesicles derived from natural killer cells use multiple cytotoxic proteins and killing mechanisms to target cancer cells
Published in Journal of Extracellular Vesicles, 2019
Chun-Hua Wu, Jingbo Li, Li Li, Jianping Sun, Muller Fabbri, Alan S. Wayne, Robert C. Seeger, Ambrose Y. Jong
Samples for immunofluorescence microscopy were prepared as follows. Neuroblastoma CHLA255 cells were plated onto glass coverslips (22 mm, square) that had been previously coated with type I collagen from rat tails (Upstate, 5–10 μg/cm2) and placed in an 8-well square culture system (Nalgene Nunc). Approximately 5 × 104 cells were seeded onto each coverslip 24 hr prior to the experiment. The mounted cells were prewashed four times with PBS, then fixed with 2% formaldehyde/PBS (v:v) for 30 min at room temperature. After three additional washes with PBS, non-specific binding was blocked with 5% milk/PBS for 30 min and cells were then incubated overnight with an antibody to cytochrome C (7H8.2C12; Biolegend; cat.#: 612503) and the MitoTracker FM dye at 4°C. The coverslips were then washed four times with PBS followed by 1% BSA/PBS. Anti-mouse IgG Rhodamine conjugate (1:100 dilution) was added into each well for 1 hr at 4°C. Another three washes were applied then a drop of Vectashield mounting solution containing DAPI (Vector Laboratories; H-120) was used to seal the coverslips onto slides. Samples were examined under a fluorescence microscope at the Congressman Dixon Cellular Imaging Core Facility of Children’s Hospital Los Angeles.
Related Knowledge Centers
- Antibody
- Biomolecule
- Dye
- Fluorescence
- Fluorophore
- Immunostaining
- Microscopy
- Epitope
- Antigen
- Fluorescence Microscope