Cryosectioning and Immunostaining of Fixed Embryos

Embryos were fixed in either 4% PFA, Dent fixative or 1% TCA for 1-3 days and subjected to cryosectioning as described previously^{31 (link)}. Antibodies used were: anti-FN antibody (Ab), Sigma F3648 at 1:100; β-integrin monoclonal Ab, 8c8 (Developmental Studies Hybridoma Bank, USA) at 1:10; anti-aPKC C-20 (SC216, Santa Cruz Biotech, USA) at 1:100; anti-PCNA (PC10, Santa Cruz Biotech, USA) at 1:500; anti-laminin (Ab-1, NeoMarkers, USA) at 1:100 and anti-ZO-1^{36 (link)} (gift from Dr M Itoh) at 1:1. Sections were counterstained with Alexa Fluor 488 or 546 Phalloidin (A12379, A22283, Invitrogen USA) at 1:250 and TO-PRO-3 (T3605, Invitrogen, USA) at 1:1000.

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.

Porazinski S., Wang H., Asaoka Y., Behrndt M., Miyamoto T., Morita H., Hata S., Sasaki T., Krens S.F., Osada Y., Asaka S., Momoi A., Linton S., Miesfeld J.B., Link B.A., Senga T., Shimizu N., Nagase H., Matsuura S., Bagby S., Kondoh H., Nishina H., Heisenberg C.P, & Furutani-Seiki M. (2015). YAP is essential for tissue tension to ensure vertebrate 3D body shape. Nature, 521(7551), 217-221.

Publication 2015

anti-FN antibody (Ab), anti-aPKC C-20 (SC216, anti-PCNA (PC10, Alexa Fluor 488 or 546 Phalloidin

Alexa fluor 488 Anti antibody Antibodies Antibody Embryos Fixative Hybridoma Integrin Laminin Laminin 1 Monoclonal antibody Pcna Phalloidin To pro 3

Corresponding Organization :

Other organizations : University of Bath, Tokyo Medical and Dental University, Institute of Science and Technology Austria, Hiroshima University, Keio University, Japan Science and Technology Agency, Medical College of Wisconsin, Nagoya University, University of Oxford, Osaka University, Kyoto Sangyo University

Top 5 similar protocols

Variable analysis

independent variables

Fixative type (4% PFA, Dent fixative, or 1% TCA)
Fixation duration (1-3 days)

dependent variables

Antibody localization/expression (FN, β-integrin, aPKC, PCNA, laminin, ZO-1)

control variables

Cryosectioning as described previously
Alexa Fluor 488 or 546 Phalloidin counterstain (1:250)
TO-PRO-3 counterstain (1:1000)

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!