scholarly journals Reduction of High Background Staining by Heating Unfixed Mouse Skeletal Muscle Tissue Sections Allows for Detection of Thermostable Antigens With Murine Monoclonal Antibodies

2008 ◽  
Vol 56 (11) ◽  
pp. 969-975 ◽  
Author(s):  
Rustam R. Mundegar ◽  
Elke Franke ◽  
Ralf Schäfer ◽  
Margit Zweyer ◽  
Anton Wernig
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibodies ◽  
Fluorescence Signal ◽  
High Background ◽  
Mouse Skeletal Muscle ◽  
Background Staining ◽  
Immunofluorescence Labeling ◽  
Secondary Antibodies ◽  
Dystrophin Protein ◽  
Murine Monoclonal Antibodies

Antigen detection with indirect immunohistochemical methods is hampered by high background staining if the primary antibody is from the same species as the examined tissue. This high background can be eliminated in unfixed cryostat sections of mouse skeletal muscle by boiling sections in PBS, and several proteins including even the low abundant dystrophin protein can then be easily detected with murine monoclonal antibodies. However, not all antigens withstand the boiling procedure. Immunoreactivity of some of these antigens can be restored by subsequent washing in Triton X-100, whereas immunoreactivity of other proteins is not restored by this detergent treatment. When such thermolabile proteins are labeled with polyclonal primary antibodies followed by dichlorotriazinylaminofluorescein-conjugated secondary antibodies and boiled, the fluorescence signal persists, and sections can then be processed with a monoclonal antibody for double immunostaining of a protein unaffected by boiling. This stability of certain fluorochromes on heating can also be exploited for double immunofluorescence labeling of two different thermostable proteins with murine monoclonal antibodies as well as for combination with Y-chromosome fluorescence in situ hybridization. Our method should extend the range of monoclonal antibodies applicable to tissues derived from the same species as the monoclonal antibodies.

scholarly journals An indirect immunofluorescence procedure for staining the same cryosection with two mouse monoclonal primary antibodies.

1993 ◽  
Vol 41 (8) ◽  
pp. 1273-1278 ◽  
Author(s):  
S A Lewis Carl ◽  
I Gillete-Ferguson ◽  
D G Ferguson
Keyword(s):  
Skeletal Muscle ◽  
Image Analysis ◽  
Monoclonal Antibodies ◽  
Indirect Immunofluorescence ◽  
Laser Scanning ◽  
Frozen Section ◽  
Laser Scanning Confocal Microscopy ◽  
Secondary Antibody ◽  
Scanning Confocal Microscopy ◽  
Secondary Antibodies

Indirect immunofluorescence procedures reported thus far are not effective at localizing two antigens in the same preparation when both primary antibodies are raised in the same species. In this case, the secondary antibodies can crossreact with both primary antibodies. We report here a protocol in which mouse monoclonal antibodies (MAb) specific for actin and myosin were used sequentially to stain the same frozen section of guinea pig skeletal muscle. The myosin-specific mAb was applied first and was localized with a rabbit anti-mouse IgG-rhodamine secondary antibody. The sections were then "blocked" with a non-binding mouse MAb and unconjugated goat anti-mouse IgG F(ab) fragments. The actin-specific mAb was then applied and localized with a rabbit anti-mouse IgG-fluorescein secondary antibody. Laser scanning confocal microscopy and image analysis demonstrated that the I-bands, the A-bands, and the H-bands of each sarcomere were clearly identifiable by this approach. This protocol is not limited to use with mouse MAb but can be easily modified to permit indirect immunolocalization of two antigens in the same sample using any pair of same-species primary antibodies.

scholarly journals A New Blocking Method for Application of Murine Monoclonal Antibody to Mouse Tissue Sections

1998 ◽  
Vol 46 (8) ◽  
pp. 977-983 ◽  
Author(s):  
Qi L. Lu ◽  
Terry A. Partridge
Keyword(s):  
Specific Antigen ◽  
Detection Methods ◽  
Signal Interference ◽  
Mouse Tissue ◽  
Mdx Mouse ◽  
Immunohistochemical Detection ◽  
Double Labeling ◽  
High Background ◽  
Background Staining ◽  
Secondary Antibodies

SUMMARY Antigen detection with primary antibody of the same species as the test tissue is complicated by high levels of background staining when indirect immunohistochemical detection methods are used. This severely limits the use of murine monoclonal antibodies on tissues of the mouse, the most widely used experimental model system; no method for blocking this is fully satisfactory. Here we show that background staining encountered in this system results largely from the binding of secondary antibodies via both Fc and Fab to endogenous immunoglobulins and other tissue components. A simple and efficient blocking strategy was established, employing papain-digested whole fragments of unlabeled secondary anti-mouse Igs enriched with Fc fragment of the same Igs. We have used this method to visualize dystrophin, an antigen expressed at low level, in revertant fibers of mdx mouse by both immunoperoxidase and immunofluorescence methods. In combination with the use of a biotin-streptavidin immunohistochemical detection protocol with biotinylated anti-mouse F(ab')2 as second layer, we eliminated the heavy background in this system and achieved strong signal amplification to demonstrate the specific antigen clearly. Double labeling with one mouse antibody and one antibody from another species was performed without signal interference. This principle can be adapted for wider applications, such as antibodies of other species on homologous tissues and perhaps where high background is found with heterologous antibodies.

scholarly journals A novel modification of the avidin-biotin complex method for immunohistochemical studies of transgenic mice with murine monoclonal antibodies.

1992 ◽  
Vol 40 (9) ◽  
pp. 1319-1328 ◽  
Author(s):  
K M Fung ◽  
A Messing ◽  
V M Lee ◽  
J Q Trojanowski
Keyword(s):  
Monoclonal Antibodies ◽  
Transgenic Mice ◽  
Detection System ◽  
Immunohistochemical Method ◽  
Mouse Tissue ◽  
Mouse Serum ◽  
Complex Method ◽  
Mouse Tissues ◽  
Secondary Antibodies ◽  
Murine Monoclonal Antibodies

When mouse tissues are probed with murine monoclonal antibodies (MAb) by indirect immunohistochemistry, the secondary antibody detects tissue-bound MAb and irrelevant, endogenous mouse immunoglobulins. The latter are a source of confounding background, especially in diseased tissues. To circumvent this problem, we generated complexes of primary MAb and biotinylated secondary antibodies in vitro for use as antigen-specific probes. After blocking free binding sites in the complexed secondary antibodies with normal mouse serum, the complexes were applied to mouse tissue sections and tissue-bound complexes were visualized with an avidin-biotin detection system. Complexes formed with 12 different rat or mouse MAb were used to probe sections of normal mice, tumor-bearing transgenic mice, and mice with tumor xenografts. The staining patterns produced by these probes reflected the specificity of the MAb in the complexes, and the labeling of irrelevant, endogenous mouse immunoglobulins was reduced substantially. This novel, indirect immunohistochemical method can be exploited to study normal and diseased mouse tissues using a variety of murine MAb.

scholarly journals The MicroRNA miR-696 is Regulated by SNARK and Reduces Mitochondrial Activity in Mouse Skeletal Muscle Through Pgc1α Inhibition

2021 ◽  
pp. 101226
Author(s):  
André L. Queiroz ◽  
Sarah J. Lessard ◽  
Amanda T. Ouchida ◽  
Hygor N. Araujo ◽  
Dawit A. Gonçalves ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Mitochondrial Activity ◽  
Mouse Skeletal Muscle

scholarly journals AS160 Regulates Insulin- and Contraction-stimulated Glucose Uptake in Mouse Skeletal Muscle*

2006 ◽  
Vol 281 (42) ◽  
pp. 31478-31485
Author(s):  
Henning F. Kramer ◽  
Carol A. Witczak ◽  
Eric B. Taylor ◽  
Nobuharu Fujii ◽  
Michael F. Hirshman ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Mouse Skeletal Muscle

scholarly journals Screening and development of monoclonal antibodies for identification of ferret T follicular helper cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Wenbo Jiang ◽  
Julius Wong ◽  
Hyon-Xhi Tan ◽  
Hannah G. Kelly ◽  
Paul G. Whitney ◽  
...  
Keyword(s):  
Animal Model ◽  
Monoclonal Antibodies ◽  
Lymph Node ◽  
Cross Reactivity ◽  
Tfh Cells ◽  
Follicular Helper ◽  
Lymph Node Cells ◽  
Human Viruses ◽  
Humoral Responses ◽  
Murine Monoclonal Antibodies

AbstractThe ferret is a key animal model for investigating the pathogenicity and transmissibility of important human viruses, and for the pre‐clinical assessment of vaccines. However, relatively little is known about the ferret immune system, due in part to a paucity of ferret‐reactive reagents. In particular, T follicular helper (Tfh) cells are critical in the generation of effective humoral responses in humans, mice and other animal models but to date it has not been possible to identify Tfh in ferrets. Here, we describe the screening and development of ferret-reactive BCL6, CXCR5 and PD-1 monoclonal antibodies. We found two commercial anti-BCL6 antibodies (clone K112-91 and clone IG191E/A8) had cross-reactivity with lymph node cells from influenza-infected ferrets. We next developed two murine monoclonal antibodies against ferret CXCR5 (clone feX5-C05) and PD-1 (clone fePD-CL1) using a single B cell PCR-based method. We were able to clearly identify Tfh cells in lymph nodes from influenza infected ferrets using these antibodies. The development of ferret Tfh marker antibodies and the identification of ferret Tfh cells will assist the evaluation of vaccine-induced Tfh responses in the ferret model and the design of novel vaccines against the infection of influenza and other viruses, including SARS-CoV2.

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