Immunostaining of Nematode Muscle Proteins

Adult and L1 larva nematodes were fixed and immunostained according to the method described by Nonet et al. (1993) (link) and described in further detail by Wilson et al. (2012a) (link). The following primary antibodies were used at 1:200 dilution: anti-UNC-89 (rabbit polyclonal EU30; Benian et al., 1996 (link)), anti-paramyosin (mouse monoclonal 5-23; Miller et al., 1983 (link)), anti–MHC A (mouse monoclonal 5-6; Miller et al., 1983 (link)), anti–MHC B (mouse monoclonal 5-8; Miller et al., 1983 (link)), anti–UNC-95 (rabbit polyclonal Benian-13; Qadota et al., 2007 (link)), anti-twitchin (rabbit polyclonal I I II; Benian et al., 1996 (link)), and anti-HA (mouse monoclonal; H3663; Sigma-Aldrich). The following antibodies were used at 1:100 dilution: anti-UNC-98 (rabbit polyclonal EU131; Mercer et al., 2003 (link)), and anti–CSN-5 (rabbit polyclonal; Miller et al., 2009 (link)). Secondary antibodies, also used at 1:200 dilution, included anti-rabbit Alexa 488 (Invitrogen, Thermo Fisher Scientific) and anti-mouse Alexa 594 (Invitrogen). Images were captured at room temperature with a Zeiss confocal system (LSM510) equipped with an Axiovert 100M microscope and an Apochromat ×63/1.4 numerical aperture oil immersion objective in ×2.5 zoom mode. The color balances of the images were adjusted by using Photoshop (Adobe, San Jose, CA).

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Qadota H., Mayans O., Matsunaga Y., McMurry J.L., Wilson K.J., Kwon G.E., Stanford R., Deehan K., Tinley T.L., Ngwa V.M, & Benian G.M. (2016). The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle. Molecular Biology of the Cell, 27(10), 1606-1620.

Publication 2016

H3663 Axiovert 100M microscope Apochromat ×63/1.4

Adult Alexa 594 Antibodies Dilution Immersion Larva Microscope Mouse Nematodes Paramyosin Rabbit Twitchin

Corresponding Organization :

Other organizations : Emory University, University of Konstanz, Kennesaw State University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Localization and expression of various muscle proteins (UNC-89, paramyosin, MHC A, MHC B, UNC-95, twitchin, UNC-98, CSN-5)

control variables

None explicitly mentioned

controls

Positive controls: None mentioned
Negative controls: None mentioned

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