Immunofluorescence Analysis of Muscle Tissue

Cells and tissues were fixed in PFA 2% and processed for histology and immunocytochemistry as previously described (Gargioli et al, 2008 (link)). The primary antibodies used were mouse anti-Pax7 (DSHB) at 1:20, mouse MF20 (DSHB) at 1:2, rabbit anti-laminin (SIGMA #9393) at 1:100, rabbit anti-LacZ (Cappel) at 1:100, rat anti-VE-cadherin (clone BV13 homemade) at 1:100, mouse anti-SMA (Sigma) at 1:100, mouse anti-dystrophin (Vector) at 1:100, mouse anti-neuronal class III β-tubulin (COVANCE) and alpha-bungarotoxin Alexa594 (Molecular probes) at 50 mg/ml. The secondary antibodies used at 1:100 were anti-mouse Alexa555 (Molecular Probes), anti-rabbit Alexa488 (Molecular Probes) and anti-rat Alexa568 (Molecular Probes), Cy2-anti-mouse (Jackson), AMCA-anti-mouse (Jackson), goat anti-mouse horseradish peroxidase (HRP)-conjugated IgG (Bio-Rad) for immunohistochemistry against MF20, developing the peroxidase reaction by AEC (3-amino-9-ethylcarbazole) substrate (SIGMA). The sections were photographed with Nikon ECLIPSE 2000-TE microscope or with Olympus FV 1000 confocal laser scanning microscope for the confocal images. VE-cad-positive capillary endothelial cells were counted under fluorescence microscopy (×200) in five randomly selected fields of different sections from each sample and related to the number of muscle fibres in the same section (Díaz-Manera et al, 2010 (link)).

Free full text: Click here

Fuoco C., Rizzi R., Biondo A., Longa E., Mascaro A., Shapira-Schweitzer K., Kossovar O., Benedetti S., Salvatori M.L., Santoleri S., Testa S., Bernardini S., Bottinelli R., Bearzi C., Cannata S.M., Seliktar D., Cossu G, & Gargioli C. (2015). In vivo generation of a mature and functional artificial skeletal muscle. EMBO Molecular Medicine, 7(4), 411-422.

Publication 2015

mouse anti-SMA anti-mouse Alexa555 anti-rat Alexa568 Cy2-anti-mouse FV 1000 confocal laser scanning microscope

3 amino 9 ethylcarbazole Alexa594 Alpha bungarotoxin Amca Anti igg Antibodies Capillary endothelial cells Cells Clone Confocal laser scanning microscope Dystrophin Fibres Fluorescence microscopy Goat Horseradish peroxidase Immunocytochemistry Immunohistochemistry Lacz Laminin Microscope Molecular probes Mouse Muscle Neuronal Pax7 Peroxidase Rabbit Tissues Tubulin Ve cadherin Vector

Corresponding Organization :

Other organizations : University of Rome Tor Vergata, MultiMedica, Institute of Cell Biology and Neurobiology, National Research Council, University of Pavia, Technion – Israel Institute of Technology, University College London, Fondazione Salvatore Maugeri, University of Manchester

Top 5 similar protocols

Protocol cited in 1 other protocol

Variable analysis

independent variables

None explicitly mentioned

dependent variables

Localization and expression of various proteins (Pax7, MF20, laminin, LacZ, VE-cadherin, SMA, dystrophin, neuronal class III β-tubulin) as determined by immunocytochemistry
Quantification of VE-cadherin-positive capillary endothelial cells per muscle fiber

control variables

None explicitly mentioned

controls

Positive controls: None specified
Negative controls: None specified

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!