Immunohistochemistry

Following transcardial perfusion with 4% paraformaldehyde (PFA) in 0.01 m PBS, brains were postfixed overnight in chilled perfusion solution before being transferred to 0.01 m PBS. 50-μm coronal slices were cut the following day on a vibratome, then washed three times and stored in 0.01 m PBS with 0.05% NaN₃ at 4°C until staining.

Slices were rinsed three times in 0.01 m PBS, then incubated in the primary antibody solution overnight (0.3% Triton X-100, 0.05% NaN₃, 1% BSA, and primary antibody in 0.01 m PBS). The following day, slices were rinsed three times in 0.01 m PBS, then incubated in the secondary antibody solution for 2 h (1% BSA and secondary antibody in 0.01 m PBS). Slices were then mounted on a glass slide with an anti-fade medium (Fluoromount-G), coverslipped, and allowed to dry overnight. Images were taken on a Leica SP5 confocal microscope. Image stacks with 488-, 543-, and 647-nm lasers were obtained using a 20× objective with an optical section height (z-step) of ∼2 μm. Images were subsequently analyzed in ImageJ.

We used the following antibodies:

anti-CaMKIV (1:1000, Santa Cruz Biotechnology sc-136249, RRID:AB_2275109),

anti-CaMKIV (1:1000, Sigma C2851, RRID:AB_258821),

anti-CaMKIV (1:1000, BD Biosciences 610275, RRID:AB_397670),

anti-pCaMKIV (1:200, Santa Cruz Biotechnology sc-28443-R, RRID:AB_2068399),

anti-VGlut2 (1:10000, Millipore AB2251, RRID:AB_1587626),

Goat anti-Mouse IgG Alexa Fluor 555 (1:300, Thermo Fisher A-21424, RRID:AB_141780),

Goat anti-Rabbit IgG Alexa Fluor 488 (1:300, Thermo Fisher A-11034, RRID:AB_2576217),

Goat anti-Guinea Pig IgG Alexa Fluor 647 (1:300, Thermo Fisher A-21450, RRID:AB_2735091).

When using the polyclonal phospho-CaMKIV (T196) antibody (sc-28443-R, Santa Cruz Biotechnology), we did not observe any difference in staining between wild-type and conditional CaMKIV knock-down mice. This suggests that the Santa Cruz Biotechnology sc-28443-R antibody, when used for immunostaining, is not specific for pCaMKIV. We attempted to immunolabel phospho-CaMKIV with antibodies obtained from multiple other sources with no success.