[

MicroPubl Biol,

2021]

The FLP-1 neuropeptide is involved in locomotion, mechanosensation, and reproduction. Strain RB2126 is reported to contain the flp-1(ok2811) mutation, a prospective null allele. Here we report that strain RB2126, in addition to flp-1(ok2811), also contains two additional background mutations. One of the mutations gives the animals a constitutive dauer phenotype (Daf-c), and complementation testing confirms that it is in the Daf-c gene daf-11. The second mutation is isolated based on the fact that it partially suppresses the elevated body curvature of flp-1(ok2811) mutants, but it remains uncloned. The updated genotype of RB2126 strain is flp-1(ok2811); daf-11(yak155); yak156.

O'Hagan R et al. (2021) Bio Protoc "Fixation and Immunostaining of Endogenous Proteins or Post-translational Modificationsin Caenorhabditis elegans."

O'Hagan R, Topalidou I

[

Bio Protoc,

2021]

Although the advent of genetically-encoded fluorescent markers, such as the green fluorescent protein (GFP; Chalfie et al., 1994 ), has enabled convenient visualization of gene expression in vivo, this method is generally not effective for detecting post-translational modifications because they are not translated from DNA sequences. Genetically-encoded, fluorescently-tagged transgene products can also be misleading for observing expression patterns because transgenes may lack endogenous regulatory DNA elements needed for precise regulation of expression that could result in over or under expression. Fluorescently-tagged proteins created by CRISPR genome editing are less prone to defective expression patterns because the loci retain endogenous DNA elements that regulate their transcription (Nance and Frokjaer-Jensen, 2019). However, even CRISPR alleles encoding heritable fluorescently-tagged protein markers can result in defects in function or localization of the gene product if the fluorescent tag obstructs or otherwise interferes with important protein interaction domains or affects the protein structure. Indirect immunofluorescence is a method for detecting endogenous gene expression or post-translational modifications without the need for transgenesis or genome editing. Here, we present a reliable protocol in which C. elegans nematodes are fixed, preserved, and permeabilized for staining with a primary antibody to bind proteins or post-translational modifications, which are then labeled with a secondary antibody conjugated to a fluorescent dye. Use of this method may be limited by the availability of (or ability to generate) a primary antibody that binds the epitope of interest in fixed animals. Thousands of animals are simultaneously subjected to a series of chemical treatments and washes in a single centrifuge tube, allowing large numbers of identically-treated stained animals to be examined. We have successfully used this protocol (O' Hagan et al., 2011 and 2017; Power et al., 2020 ) to preserve and detect post-translational modifications of tubulin in C. elegans ciliated sensory neurons and to detect non-modified endogenous protein (Topalidou and Chalfie, 2011).