The study was carried out on tissues dissected from 4 adult, 1 male and 3 female, Wistar rats (weight 140–270 g; age 7–16 wk), handled in accord with the Guiding Principles for Care and Use of Animals of the Physiological Society of Japan. The rats were deeply anesthetized with diethyl ether, and after opening the chest were instantly perfused through the heart with 4% paraformaldehyde in phosphate buffered saline (PBS), pH 7.20. The carotid bodies were dissected bilaterally from the carotid bifurcation area. The nearby sympathetic superior cervical ganglions also were dissected to be used as reference specimens for both CaMKs and tyrosine hydroxylase (TH), a marker of chemoreceptor cells. Additionally, the brain was extracted from the skull and the hippocampus was cut out for positive CaMKs controls. All the tissues were immersed in the same fixative in which they were postfixed for 1.5 h and then cryoprotected in 30% sucrose at 4°C until further use.
Sections of the carotid body (10 μm thick), superior cervical ganglion (15 μm thick), and hippocampal (30 μm thick) tissues were made (CM1900 cryostat, Leica Instruments, Nussloch, Germany) and attached to glass slides coated with poly-L-lysine. Sections from all the tissues were processed on the same glass slide for a given CaMK.
The immunohistochemical procedures were performed in room temperature and consisted of five successive steps. I – solubilization of specimens in 0.3% Triton X-100 in PBS for 15 min; II – blocking the nonspecific binding with 5% normal goat serum for 30 min; III – overnight incubation with primary rabbit IgG rabbit antibodies against CaMKI, CaMKII, and CaMKIV (dilution 1:1000, 1:1000, and 1 3 μg/ml, respectively) and with a PCTH-7 mouse monoclonal IgG antibody against TH for double staining to confirm the chemoreceptor cell localization of immunostaining; IV – 1 h incubation with secondary goat Alexa Fluor 488-conjugated antirabbit + goat Alexa Fluor 594-conjugated antimouse antibodies diluted at 1:2000 (Alexa Flour; Molecular Probes, Eugene, OR) in the environment protected from light; and V – nuclear staining with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO). Steps III, IV, and V were followed by several sequential washes of specimens in PBS. Finally, the specimens were covered with an embedding medium and examined by visual inspection and photographed under a confocal laser scanning microscope and workstation (LSM 5 Pascal; Carl Zeiss, Jena, Germany).
Antibodies against CaMKI were produced in-house. Briefly, the entire coding region of rat CaMKIα was amplified by PCR and inserted in frame to pGEX4T-1 expression vector (Amersham Biosciences, Piscataway, NJ). The glutathione S-transferase (GST)-CaMKIα fusion protein was induced in E. coli in the presence of isopropyl-1-thio-β-D-galactoside. The fusion protein was then purified from a soluble extract by glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s protocol. Two female New Zealand rabbits were immunized by repeated intradermal injections of the fusion protein emulsified with an equal volume of Freund's adjuvant. The antibody against CaMKI was purified by a protein A-sepharose 4B column, followed by the A-sepharose 4B column coupled with GST-CaMKIα. Western blots showed that the antibody reacted with CaMKIα and δ, but not β2 and γ1 in a single band. Antibodies against CaMKIV were made according to the method described previously (Sakagami et al. ) and those against CaMKII were generously provided by Prof. Kohji Fukunaga of Tohoku University Graduate School of Pharmaceutical Sciences.
Positive controls showed the CaMKs immunostainings in the hippocampus, which was particularly relevant regarding the CaMKIV that appeared as streaks of stained neuronal nuclei. The CaMKs and TH closely overlapped in the sympathetic neurons of the superior cervical ganglion, as is reported in the literature (Arciszewski et al. ). Negative controls, in which the primary antibody was omitted, failed to express immunostaining. Thus, both positive and negative controls (not shown) verified the soundness of the immunostaining.