Immunofluorescence Characterization of Cell-Matrix Adhesions

Cultures were fixed with 4% paraformaldehyde (2D: 20 minutes, room temperature; 3D: overnight, 4°C) and staining was performed as described (1 (link)). Primary antibodies against vinculin (hVIN-1, Sigma Aldrich; 700062, Invitrogen; V284, Santa Cruz Biotechnology), β1 integrin (AIIB2, isolated from rat hybridoma), β4 integrin (3E1, Millipore MAB1964), ^p397FAK (44625G,Invitrogen), ^p473Akt (Cell Signaling 9271S), Akt(pan)-Alexa488 (Cell Signaling C67E7), E-cadherin (610181; BD Transduction),β-catenin (610153, BD), pan-laminin (L9393, Sigma), laminin 5 (P3H9, isolated from mouse hybridoma), α6 integrin (GoH3, eBiosciences), phospho-myosin light chain kinase 2 - Thr18/Ser19 (3674, Cell Signaling), talin (T3287, Sigma), zyxin (BD, 610521), and AlexaFluor phalloidin (633-conjugate, Invitrogen) were used. Secondary antibodies used include AlexaFluor goat anti-mouse, anti-rabbit, and anti-rat (488, 568 and 633 conjugates).

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Rubashkin M., Cassereau L., Bainer R., DuFort C., Yui Y., Ou G., Paszek M., Davidson M., Chen Y, & Weaver V. (2014). Force engages vinculin and promotes tumor progression by enhancing PI3-kinase activation of phosphatidylinositol (3,4,5)-triphosphate. Cancer research, 74(17), 4597-4611.

Publication 2014

hVIN-1 p473Akt E-cadherin β-catenin talin AlexaFluor phalloidin

Antibodies E cadherin Goat Hybridoma Integrin Laminin Laminin 5 Mouse Myosin light chain kinase Paraformaldehyde Phalloidin Rabbit Talin Vinculin Zyxin β catenin β1 integrin

Corresponding Organization : University of California, San Francisco

Other organizations : Cornell University, National High Magnetic Field Laboratory, Florida State University

Top 5 similar protocols

Protocol cited in 2 other protocols

Variable analysis

independent variables

Fixation time (2D: 20 minutes, room temperature; 3D: overnight, 4°C)

dependent variables

Staining for vinculin, β1 integrin, β4 integrin, p397FAK, p473Akt, Akt(pan)-Alexa488, E-cadherin, β-catenin, pan-laminin, laminin 5, α6 integrin, phospho-myosin light chain kinase 2 - Thr18/Ser19, talin, and zyxin

control variables

Fixation protocol
Primary antibodies used
Secondary antibodies used

controls

Positive control: Not specified
Negative control: Not 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!