Photomicrographs of mouse SCN sections immunostained with BMAL1 and CLOCK antibodies.

<p>The left column shows sections from wild-type mice euthanized at ZT12. The center column shows sections from wild-type mice euthanized at ZT0. The right column shows sections from mice lacking the target antigen (“mutant") at ZT0 or 12, as indicated. The antigen, host number (R = rabbit; GP = guinea pig) and antibody concentration used for each antibody is indicated. Scale bar, 100 µm.</p

Summary of Immunostaining Results in Hamsters.

<p>Host #: R = rabbit; GP = guinea pig. Peak Time: Hamsters were perfused at the time of expected peak of immunostaining and a series of dilutions was examined. Nadir Time: Hamsters were perfused at the expected low point of immunostaining rhythms (ZT0 for PER1, PER2, and ZT12 for BMAL1). CLOCK immunostaining was comparable at ZT0 and ZT12; dilution studies were conducted at ZT12 and results from ZT0 are listed arbitrarily as Nadir Time. Studies at nadir time used the antibody concentration found to give optimal labeling at the peak time. N = number of animals tested. Results are scored as Described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035938#pone-0035938-t001" target="_blank">Table 1:</a> −(no SCN cells labeled), +(relatively poor staining), ++(good staining in SCN), and +++(best staining of SCN cells compared to background). NS indicates non-specific staining (cells inside and outside the SCN were labeled with the same intensity). “n.d." indicates not determined.</p

Photomicrographs of hamster SCN sections immunostained with PER1 antibodies.

<p>PER1 antibodies R1177, R43 and GP91 were used at a concentration of 1∶5,000. Animals were euthanized at ZT12. Scale bar, 100 µm.</p

Photomicrographs of mouse SCN sections immunostained with PER1 and PER2 antibodies.

<p>The left column shows sections from wild-type mice euthanized at ZT12. The center column shows sections from wild-type mice euthanized at ZT0. The right column shows sections from mice lacking the target antigen (“mutant") at ZT12. The antigen, host number (R = rabbit; GP = guinea pig) and antibody concentration used for each antibody is indicated. Scale bar, 100 µm.</p

Photomicrographs of hamster SCN sections immunostained with PER1, PER2, BMAL1 and CLOCK antibodies.

<p>Columns show the staining at ZT12 (left) and ZT0 (right) level for each antigen. The antigen, host number (R = rabbit; GP = guinea pig) and antibody concentration used for each antibody is indicated. Scale bar, 100 µm.</p

Summary of Immunostaining Results in Wild-type and Mutant Mice.

<p>Host #: R = rabbit; GP = guinea pig. Wild-type mice were perfused at the time of expected peak of immunostaining (ZT12 for PER1 and PER2; ZT0 for BMAL1), and at the expected low point (nadir) of immunostaining rhythms (ZT0 for PER1, PER2, and ZT12 for BMAL1). Mutant mice were examined at the time of peak staining in wild-type mice. Results from wild-type mice at peak time are reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035938#pone-0035938-t001" target="_blank">Table 1</a>, for comparison. CLOCK immunostaining was comparable at ZT12 (arbitrarily called Peak Time) and ZT0 (arbitrarily called Nadir Time). CLOCK-deficient mice were examined at ZT12. N = number of animals tested. Results are scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035938#pone-0035938-t001" target="_blank">Table 1:</a> −(no SCN cells labeled), +(relatively poor staining), ++(good staining in SCN), and +++(best staining of SCN cells compared to background). NS indicates non-specific staining (cells inside and outside the SCN were labeled with the same intensity).</p

Assessment of Antibody Dilutions in Wild-Type Mice.

<p>Host #: R = rabbit; GP = guinea pig. Peak Time: Wild-type mice were perfused at the time of expected peak of immunostaining and a series of dilutions was examined. N = number of animals tested. Results are scored as: −(no SCN cells labeled), +(relatively poor staining), ++(good staining in SCN), and +++(best staining of SCN cells compared to background). NS indicates non-specific staining (cells inside and outside the SCN were labeled with the same intensity). “n.d." indicates not determined.</p