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were isolated from 21-d-old mice, perfused with 4% paraformaldehyde in PBS, and post-fixed overnight in the same fixative. Brains were embedded in gelatin, and 35-μm sagittal sections were cut using a sliding microtome (NeuroScience Associates, http://www.neuroscienceassociates.com). Sections from wild-type, heterozygote, and homozygous brains were placed in the MultiBrain template. Sections were washed in 1× PBS prior to 1 h of incubation in block solution containing 1× PBS with 20% normal goat serum and 0.3% Triton X-100 (pH 7.4). Sections were incubated overnight at 4 °C in primary antibodies: affinity purified polyclonal Itpr1 antibody (1:2,000, Chemicon International, http://www.chemicon.com) and monoclonal anti-Calb1 antibody (1:6,000, Sigma-Aldrich, http://www.sigmaaldrich.com) diluted in carrier solution. Following extensive washes (in 6.0 ml of PBS, three times), sections were incubated with appropriate secondary antibodies (Alexa Fluor 555 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG [Invitrogen, http://www.invitrogen.com]) for 1 h at room temperature. Sections were washed and mounted on glass slides in a buffered medium containing Mowiol (Calbiochem, http://www.emdbiosciences.com) as described earlier [14]. Sections were imaged using a laser scanning confocal microscope (LSM 510; Zeiss, http://www.zeiss.com). Imaging parameters (pinhole, detector gain, laser power) were optimized, and were kept constant for the wild-type, heterozygous, and homozygous mutant brains. Specificity of the Itpr1 antibodies was verified by preabsorption control experiments. Antibody dilutions were incubated for 24 h at 4 °C with the immunizing peptide. Tissue sections were incubated with the preabsorbed antibodies and processed as described above. Under these conditions, no staining above autofluorescence was detected."}

{"project":"2_test","denotations":[{"id":"17590087-12112377-85657861","span":{"begin":1278,"end":1280},"obj":"12112377"},{"id":"T31713","span":{"begin":1278,"end":1280},"obj":"12112377"}],"text":"Immunohistochemistry.\nBrains were isolated from 21-d-old mice, perfused with 4% paraformaldehyde in PBS, and post-fixed overnight in the same fixative. Brains were embedded in gelatin, and 35-μm sagittal sections were cut using a sliding microtome (NeuroScience Associates, http://www.neuroscienceassociates.com). Sections from wild-type, heterozygote, and homozygous brains were placed in the MultiBrain template. Sections were washed in 1× PBS prior to 1 h of incubation in block solution containing 1× PBS with 20% normal goat serum and 0.3% Triton X-100 (pH 7.4). Sections were incubated overnight at 4 °C in primary antibodies: affinity purified polyclonal Itpr1 antibody (1:2,000, Chemicon International, http://www.chemicon.com) and monoclonal anti-Calb1 antibody (1:6,000, Sigma-Aldrich, http://www.sigmaaldrich.com) diluted in carrier solution. Following extensive washes (in 6.0 ml of PBS, three times), sections were incubated with appropriate secondary antibodies (Alexa Fluor 555 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG [Invitrogen, http://www.invitrogen.com]) for 1 h at room temperature. Sections were washed and mounted on glass slides in a buffered medium containing Mowiol (Calbiochem, http://www.emdbiosciences.com) as described earlier [14]. Sections were imaged using a laser scanning confocal microscope (LSM 510; Zeiss, http://www.zeiss.com). Imaging parameters (pinhole, detector gain, laser power) were optimized, and were kept constant for the wild-type, heterozygous, and homozygous mutant brains. Specificity of the Itpr1 antibodies was verified by preabsorption control experiments. Antibody dilutions were incubated for 24 h at 4 °C with the immunizing peptide. Tissue sections were incubated with the preabsorbed antibodies and processed as described above. Under these conditions, no staining above autofluorescence was detected."}