Date Published: April 27, 2012
Publisher: Public Library of Science
Author(s): Joseph LeSauter, Christopher M. Lambert, Margaret R. Robotham, Zina Model, Rae Silver, David R. Weaver, Shin Yamazaki. http://doi.org/10.1371/journal.pone.0035938
Research on the mechanisms underlying circadian rhythmicity and the response of brain and body clocks to environmental and physiological challenges requires assessing levels of circadian clock proteins. Too often, however, it is difficult to acquire antibodies that specifically and reliably label these proteins. Many of these antibodies also lack appropriate validation. The goal of this project was to generate and characterize antibodies against several circadian clock proteins. We examined mice and hamsters at peak and trough times of clock protein expression in the suprachiasmatic nucleus (SCN). In addition, we confirmed specificity by testing the antibodies on mice with targeted disruption of the relevant genes. Our results identify antibodies against PER1, PER2, BMAL1 and CLOCK that are useful for assessing circadian clock proteins in the SCN by immunocytochemistry.
The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus generates daily rhythms in behavior, hormones and physiology. The SCN is composed of a heterogeneous population of neurons which form a functional circadian clock as a result of coupling through neurochemical interactions , . Individual cells from SCN and from many other tissues express 24-hour molecular rhythmicity that results from a transcriptional-translational feedback loop. The transcription factors, CLOCK and BMAL1, form heterodimers and bind to E-box elements in the promoters of Period (Per) 1 and Per2, Cryptochrome (Cry) 1 and Cry2 genes. The protein products of these genes form complexes, which translocate into the nucleus and interact with the CLOCK/BMAL1 complex, resulting in repression of their transactivational activity , . Post-translational events modify the timing of this negative feedback, providing fine control over cycle length of the molecular oscillations –.
Table 1 provides a list of all antibodies tested and the results of dilution series conducted at times when levels of the corresponding antigen were expected to be high in wild-type mice. Table 2 lists the results of studies conducted at the optimal dilution, comparing results at expected peak and nadir times, and the results of mutant mice examined at the peak time. Table 3 summarizes results from studies examining hamster SCN. Figures 1, 2, and 3 show photomicrographs of SCN staining for those antibodies producing strong SCN staining intensity at the concentrations that give the best labeling. Results for antibodies with poor staining are not shown in the figures.
The lack of good antibodies makes it difficult to assess the impact of circadian time, phase shifting stimuli, or clock gene mutations on clock gene protein products. While there are commercially available antibodies, many have not been subjected to validation to assess their usefulness in immunocytochemistry. Here we generated several antibodies against the clock proteins PER1, PER2, BMAL1 and CLOCK, and tested them for staining in the SCN of both mice and hamsters (Tables 1, 2, and 3), identifying several that gave excellent results (Figs. 1, 2, and 3).