In the nematode satellite model organism Pristionchus pacificus, transgenics and CRISPR/Cas9 have been implemented in the last decade (Schlager et al., 2009; Cinkornpumin & Hong, 2011; Witte et al., 2015; Namai & Sugimoto, 2018). While CRISPR has been used to make mutants and to knock-in short sequences, sequence insertion to date is limited to ~150 base pairs. Therefore, following expression from native loci in P. pacificus currently depends on epitope-tagging the gene of interest via CRISPR and template-dependent repair, and then using immunocytochemistry with its maddening, nearly infinite possible variations in fixation, permeabilization, blocking, and antibody incubation protocols.
We used CRISPR/Cas9 to epitope-tag P. pacificus orthologs of two transcription factor genes known to function in neuronal specification in C. elegans:
unc-3 (Prasad et al., 1998; Kratsios et al., 2011) and
unc-86 (Finney and Ruvkun, 1990; Zhang et al., 2014).
Ppa-unc-3 was tagged at the C-terminus with 2x-FLAG; a single FLAG tag was added to the C-terminus of
Ppa-unc-86. Using an Improved Finney-Ruvkun-style fixation, permeabilization and antibody incubation method (similar to Bettinger et al., 1996; Finney & Ruvkun, 1990; and Peroxide tube fixation described in Duerr, 2006), we observed strong nuclear staining in neurons in four independent
Ppa-unc-3::2xFLAG strains, although the staining was highly variable both within and between preps (and worm stages), and only a small subset of worms stained well. On the contrary, in three independent
Ppa-unc-86::1xFLAG strains, we never saw any nuclear staining. After repeated attempts with some variations proved unsuccessful, we included a treatment long-known and widely-used with formalin-fixed and paraffin-embedded tissues in the field of pathology (Shi et al., 1990; reviewed by Yamashita, 2007), but apparently neglected in the worm field: heat-induced antigen retrieval (HIAR), also known as heat-induced epitope retrieval (HIER). In this method, after fixation, tissue is heated to boiling temperature (or higher, under pressure), typically for a short time (~60 min or less) using boiling water, microwave, autoclave, pressure cooker, etc. Likely the main mechanism of HIAR is breaking covalent bonds between proteins and other components formed during fixation, making antigens accessible by unmasking and changing their conformation, and perhaps also enhancing overall permeability of the tissue. HIAR is highly pH-dependent, working in low (<4) or high (>8) pH solutions, with high pH generally preferred for better tissue preservation (see review by Yamashita, 2007). In one study, HIAR was found to be essential for antibody staining against all 9 tested nuclear antigens, and 7 of 26 cytoplasmic and cell membrane antigens in ethanol-fixed human tissues (Denda et al., 2012).
We treated fixed P. pacificus worms in BO3 (borate) buffer [10 mM H3BO3, 10 mM NaOH, 1% Triton X-100, pH ~9] in an Eppendorf Thermomixer block at 99C (maximal setting) for 15 or 30 min, prior to incubation/blocking in protein-containing Antibody Buffer B solution (see below). In the case of
unc-3::2xFLAG strains, both 15 and 30 minute heat treatments dramatically improved the staining greatly reducing variability, with almost all animals staining moderately well to very well (Fig. 1A). For the
unc-86::1xFLAG strain, for the first time we saw nuclear staining in many worms (Fig. 1B). (The expression patterns of both
Ppa-unc-3::2xFLAG and
Ppa-unc-86::1xFLAG strains will be described elsewhere. It should also be noted that both a mouse anti-FLAG monoclonal and rabbit anti-FLAG polyclonal antibody recognized an epitope in wild type P. pacificus worms that appears to mark cell junctions, especially in the intestine. This does not typically interfere with identification of neuronal nuclei in the strains described.) One disadvantage to the HIAR treatment in our preparations is an increased variability and reduction or loss in DAPI staining, with high background. Normally, this fixation and permeabilization method (minus HIAR) yields uniformly excellent DAPI staining. With HIAR, in some cases, we observed the reduction or loss of staining to be selective: smaller, more compact nuclei such as those of sperm and neurons retained their DAPI staining better than others (e.g., hypodermal and intestinal nuclei). A shorter HIAR treatment (15 min) appears to preserve DAPI staining better. In a separate experiment, a 5 min HIAR treatment was insufficient to reveal more than a trace of
Ppa-unc-86::1xFLAG staining compared to 15 and 30 min treatments, although DAPI staining was better preserved.
In conclusion, if antibody staining experiments with conventional protocols are not working well, we suggest adding HIAR to the end of a standard protocol prior to blocking and antibody incubation. As a potential caveat, we note it is possible that this works well only for antigens like the FLAG epitope, and/or for these nuclear antigens, and not with others. We have not yet tried HIAR with other antigens or other fixation methods.