Antibody News

Multiple fluorescent labeling with secondary antibodies for immunocytochemistry and immunohistochemistry is a powerful tool to examine the behavior and interactions of more than one protein in a cell or tissue sample.  However, there are a few guidelines to follow to make sure your samples are correctly labeled. Read our top five tips for a successful multiple antibody labeling experiment:

1. Select primary antibodies raised in different host species. If you must use antibodies from the same host, use different IgG isotypes. Perform single staining of the antibodies prior to introducing multiple antibodies and secondary antibodies to the experiment, this will allow you to understand their isolated behavior and expression.
   
   
2. When selecting secondary antibodies, make sure they come from the same host species, but have different fluorophores within a safe range of spectra to avoid overlap. It is a...

Flow cytometry is an experimental method that was developed to label and examine a high volume of cells in an extremely rapid rate using antibodies conjugated to fluorophores.  The basic concept of flow cytometry is that a cell suspension is situated into a single stream, which then passes through a light source that uses detectors to generate data sets based off cellular properties.  More specifically, the fluorescent light emitted by fluorophores conjugated to antibodies is channeled through selected filters to sort based off preset parameters or targets used. Flow cytometry is particularly useful in cell viability and proliferation assays, as well as diagnosing disease (particularly blood cancers).  When creating a panel of fluorophores for your flow cytometry panel, it is important to follow a few basic guidelines.

First, it is important to understand your flow cytometer.  You should know the type of laser in your instrument, the number of lasers present and...

Stimulator of interferon genes (STING), also known as TMEM173, promotes the production of the interferon’s IFN-alpha and IFN-beta.  STING possesses three functional domains: a cytoplasmic C-terminal tail, a central globular domain, and four N-terminal transmembrane motifs that attach it to the ER.  The role of STING in the immune response is specific to its ability to sense nucleic acids, particularly dsDNA.  When STING is over induced, the protein IRF3 undergoes a nuclear translocation resulting in IFN induction, which in turn activates the innate immune response. The herpes virus is a DNA based virus targeted to DNA-sensing pathways that has the capacity to elicit latent recurrent infections on a host. Because of the overlap between the herpes virus affecting DNA-sensing and the ability of STING to sense nucleic acids, using...

Immunocytochemistry/immunofluorescence (ICC/IF) involves visualization of antigens in cultured or smeared cells with the use of fluorochrome-labeled antibodies. Various combinations of fluorochrome-labeled antibodies make it possible to simultaneously detect multiple antigens in the same sample. However, every experiment involving multiplex or multicolor ICC/IF requires selection of an optimal set of fluorochromes. Poor selection of fluorochrome combinations can make it difficult to distinguish individual antigens, especially when the targets under consideration are co-localized at the sub-cellular level. Here we have outlined key rules for selecting fluorochromes to minimize potential errors and to simplify the selection process.

1. Check for fluorochrome(s) compatibility with your microscope’s filter sets

Use spectra viewers and fluorochrome reference charts to determine the maximum excitation (Ex) and emission (Em) wavelengths...

Traumatic brain injury (TBI) currently contributes to nearly 30% of all injury deaths in the United States.  Characterized by an abrasive head injury that interrupts normal brain function, TBI can range from mild to severe.  Mild symptoms can present themselves as excessive tiredness, difficulty concentrating and lack of clear thinking.  Severe cases of TBI are hallmarked by unusual behavior, seizures and loss of consciousness.  Research has shown that on a molecular level TBI triggers various mechanisms of cell death alongside attempted tissue recovery, therefore Choi et al sought out to test the treatment of carbon monoxide on rescuing the effects of TBI.  Their article titled “Dual effects of carbon monoxide on pericytes and neurogenesis in traumatic brain injury” tests the effects of CO-releasing molecular 3 (CORM-3) and N-tert-butyl-a-phenylnitrone (PBN) on a mouse model of controlled cortical impact TBI.

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Have you ever wondered why cells in the same population respond differently to an apoptotic stimulus? Apoptosis, a form of programmed cell death, is vital for the removal of unwanted or damaged cells. As with most cellular processes, too much or too little activation can be detrimental and lead to various diseases including autoimmune disorders and cancer. While this process is tightly regulated, cells undergo apoptosis in a non-synchronized manner, which complicates the analysis of apoptotic events.  Here are 3 keys factors that determine how an individual cell will respond to an apoptotic stimulus.

Processed caspase 3 was detected in immersion fixed anti-FAS treated Jurkat human acute T cell leukemia cell line using Human/Mouse Active Caspase 3 Polyclonal Antibody (...

The major histocompatibility complex (MHC) is responsible for binding peptide fragments arising from pathogens in order to display them on the cell surface for recognition from immune cells.  Once recognized, the foreign pathogen is typically evaded. The MHC complex is broken into two categories, MHC Class I proteins and MHC Class II proteins.  MHC complex I and II proteins are all very different and contain specific molecules to bind different peptides – in fact, they have been described as the most polymorphic genes there are. The MHC Class II RT1B antibody can be used to bind the monomorphic determinant of the rat I-A antigen, which is found on B-lymphocytes, dendritic cells and some macrophages. Often times, a neuroinflammatory response develops after brain injury and remains for weeks post injury.  Using a MHC Class II RT1B antibody is a useful way to understand...

c-Fos is a member of the AP-1 transcription factor family under the Fos protein family umbrella, alongside Fra-1, Fra-2 and Fos-B.  Also in the AP-1 transcription family are the Jun proteins, c-Jun, Jun-B and Jun-D.  Each member of the AP-1 transcription family is a phosphonuclear protein composed of a carboxy-terminal leucine zipper domain, a basic domain and an amino terminal transactivation domain. Together the Fos and Jun families compose a dimeric complex that binds to response elements on DNA in order to regulate gene expression, cell proliferation, cell differentiation, tumorigenesis and more.  Their induction can be catalyzed by a number of signals, including cytokines and growth factors, stress and viral infection. ...

Immunocytochemistry (ICC) can be a very effective method for visualizing the localization and behavior of intracellular proteins, however the protocol for each ICC experiment should be optimized specifically to the cell being examined.  Permeabilization, or the puncturing of the cell membrane, is an extremely important step in detecting intracellular antigens with a primary antibody because it allows entry through the cell membrane. Permeabilization is introduced after cells have been prepared with a fixative agent to initiate protein cross-linking, such as formaldehyde or ethanol. However, determining the amount of exposure to a permeabilization agent is crucial, seeing as a hard tissue organ may require a longer incubation over a soft tissue organ and treating your sample incorrectly will lead to unreliable results. 

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Cell death/recycling pathways such as apoptosis, necroptosis, and autophagy are an integral part of the growth, development, homeostasis as well as the pathophysiology in the life of living organisms. These signaling pathways are highly regulated and some of their key regulatory targets are discussed below.

Apoptosis

Apoptosis, programmed cell death, is primarily characterized by the activation of caspases which further regulate the mass cleavage of proteins and DNA. Some of major the proteins responsible for various apoptotic events are:

Mammalian target of Rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, proliferation, motility and survival. Acting downstream of AKT, also a serine kinase, mTOR is composed of the mTORC1-Raptor complex and the mTORC2-Rictor complex.  mTOR has been implicated in many cancers, including ovarian cancer, due to the effect of mTOR inhibitors on tumor progression.  In addition, mTORC2 has a positive feedback effect on AKT behavior, which may explain its rapamycin resistance.  Rapamycin is a common drug used as a targeted therapy for mTOR inhibition, however it is not successful in all cancer types. In Ovarian Cancer specifically, the PI3K/AKT/mTOR pathway is the most frequently altered pathway, with PI3K, AKT and mTOR expression levels correlating with survival and tumor growth.  In the following articles, an mTOR antibody is used to investigate the efficacy of two...

Signal amplification methods are widely used in immunohistochemistry (IHC) for detection of rare epitopes and low abundance antigens. While many of these techniques such as the avidin-biotin complex (ABC) method improve staining, they frequently require additional steps and result in higher background staining.  Blocking endogenous biotin, a requirement of using ABC reagents, may not sufficiently remove residual activity in frozen tissue sections and tissues high in biotin including the liver and kidney.

Figure 1. FABP1/L-FABP was detected in paraffin-embedded sections of human kidney using Mouse Anti-Human FABP1/L-FABP Monoclonal Antibody (Catalog # MAB2964) at 1 μg/mL for 1 hour at room temperature followed by incubation for 30 minutes at room temperature with Anti-Mouse/Rabbit IgG VisUCyte HRP Polymer Antibody (Catalog...