Immunofluorescent Localization of Proteins in Caenorhabditis elegans Muscle

Abstract

Summary C. elegans is a premier model genetic system for discovering new information about the assembly and maintenance of striated muscle. The localization of a protein within a nematode muscle cell can reveal important clues to its function. In C. elegans, proteins can be localized by two different methods at the light microscopy level: GFP tagged proteins and indirect immunofluorescence. Although there are advantages and disadvantages of each method, antibodies can be used to localize proteins expressed at endogenous levels and without tags that might interfere with function. Immunolocalization requires efficient and effective methods of fixation. Here, we describe in detail two different methods for fixation of adult worms, the Nonet method and the Constant Spring method. We also discuss the advantages and disadvantages of each, and how to choose between them. These methods are also useful for localizing proteins expressed in other cell types.

High‐resolution imaging of muscle attachment structures in Caenorhabditis elegans

“…Adult nematodes were fixed and immunostained according to the method described [Nonet et al, 1993], and described in further detail [Wilson et al, 2012]. The following primary antibodies were used at 1:200 dilution: anti-UNC-89 (mouse monoclonal MH42 [Benian et al, 1996; Hresko et al, 1994]); anti-UNC-89 (rabbit polyclonal EU30 [Benian et al, 1996]); anti-myosin heavy chain A (MHC A) (mouse monoclonal 5–6; [Miller et al, 1983]), anti-myosin heavy chain B (MHC B) (mouse monoclonal 5–8; [Miller et al, 1983]), anti-UNC-52 (mouse monoclonal MH2; [Mullen et al, 1999]), anti-DEB-1 (mouse monoclonal MH24; [Francis and Waterston, 1991]), anti-ATN-1 (mouse monoclonal MH35; [Francis and Waterston, 1991]), and at 1:100 dilution: anti-UNC-95 (rabbit polyclonal Benian-13; [Qadota et al, 2007]), anti-UNC-112 (rabbit polyclonal; [Hikita et al, 2005]), anti-PAT-4 (rabbit polyclonal; [Qadota et al, 2012]), anti-UNC-97 (rabbit polyclonal; [Miller et al, 2006]), anti-PAT-6 (rat polyclonal; [Warner et al, 2013]), anti-UNC-98 (rabbit polyclonal; [Mercer et al, 2003]), anti-ALP-1 (rabbit polyclonal; [Han and Beckerle, 2009]), anti-MIG-6 (rabbit polyclonal; [Kawano et al, 2009]).…”

“…Adult nematodes were fixed and immunostained according to the method described in Nonet, Grundahl, Meyer, and Rand (1993) and described in further detail in Wilson, Qadota and Benian (2012). The following primary antibodies were used at 1:200 dilution: anti-UNC-89…”

Suppressor Mutations Suggest a Surface on PAT-4 (Integrin-linked Kinase) That Interacts with UNC-112 (Kindlin)

“…Immunostaining-Nematodes were fixed using the method described previously (28,29). Antibody staining with anti-HA (Sigma Aldrich H3663, 1/200 dilution), anti-GFP (Invitrogen A11122, 1/200 dilution) and anti-UNC-95 (1/100 dilution) were performed as described previously (30).…”

A Region of UNC-89 (obscurin) Lying Between Two Protein Kinase Domains is a Highly Elastic Spring Required for Proper Sarcomere Assembly

“…Adult worms were fixed as described previously [55,56]. For body wall muscle straining, these primary antibodies were used at the following dilutions: anti-myosin heavy chain A…”