This study was based on data from 28 adult Gekko gecko of both sexes. All animal care and anesthesia procedures followed the procedures approved by the University of Maryland College Park Animal Care And Use Committee. Geckos were anesthetized in a mixture of isofluorane and room air in a small chamber, followed by i.p. injection of euthasol at a dose of 7 mg/kg. Once the geckos were deeply anesthetized (no response to toe pinch, depressed respiration), they were perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer for 1 hour. The brains were postfixed in the fixative overnight at 4°C and cryo-protected in 30% sucrose in 0.1 M phosphate buffer at 4°C. The brains were sectioned on a freezing microtome at 40 μm thickness, and all the sections were collected in order in phosphate buffer. Most brains were cut in the coronal plane, with three brains cut in the horizontal and two in the sagittal plane.
Immunohistochemical procedures used the avidin-biotin-peroxidase complex (Vector Laboratories) method in conjunction with horseradish peroxidase substrate reagent SG kits (Vector Laboratories, Burlingame, CA). Free-floating sections were preincubated for 1 hour in a blocking solution of 10% normal goat serum diluted in 0.1 M phosphate-buffered saline (pH 7.4) containing 0.3% Triton X-100. Subsequently, sections were incubated with antisera against 1) CR (7699/66; Swant, Belinzona, Switzerland) diluted in 1:5,000; 2) PV (PA-235; Sigma, St. Louis, MO) diluted 1:2,000; 3) CB (300; Swant) diluted in 1:3,000; 4) GAD (G5163; Sigma) diluted 1:1,000; 5) SV2 (Hybridoma Bank, University of Iowa) diluted in 1:1,000 for 2 days at 4°C (). After multiple washes, sections were incubated for 1 hour in biotinylated goat anti-rabbit (for GAD, CR) or goat anti-mouse (for PV, CB, SV2) secondary antisera, diluted 1:500. Sections were incubated in ABC, followed by a horseradish peroxidase reaction. Sections were then mounted, dried, dehydrated, and coverslipped. Some sections were additionally counter-stained with neutral red.
Cerebellar tissue was used to optimize staining patterns. It was incubated in the presence or absence of primary antibodies, and, in their absence, immunostaining of Purkinje cells and granule cells was eliminated. By contrast, the Purkinje cells heavily expressed CB and PV but not CR, whereas CR labeled stellate cells (Bastianelli, 2003
). Serial dilution controls were used to optimize the working concentration of each antibody. Four serial dilutions of primary antibody ranged from 1:500 to 1:16,000, and sections were processed as described above, shared common reaction baths, and were incubated simultaneously. We measured the optical density of labeled neurons with respect to antibody dilution, and all antibody concentrations used were in the linear stages of the optical density vs. concentration plot. The dilution used for PV (1:2,000) was at the high end of the linear range because of light staining for PV in gecko (). To double-label CR and PV or CB, sections were incubated in two primary antisera simultaneously. After washing, sections were incubated in a mixture of two secondary antibodies, Alexa 594 donkey anti-rabbit and Alexa 488 donkey anti-mouse (Invitrogen, Carlsbad, CA) in the dark at a dilution of 1:500. The labeled sections were coverslipped with an antifade kit (Invitrogen), and images were obtained with a confocal microscope (Zeiss LSM 510). Digital images of selected sections were captured either on the confocal microscope or with a digital camera (DVC, Austin, TX) mounted on an Olympus BX-60 microscope. Image contrast adjustments and photomontages were performed in Adobe Photoshop 11 (Adobe Systems, Mountain View, CA).
Western blots for CBPs, GAD, and SV2 in gecko brain. All blots except that for SV2 yield a single band at estimated molecular weights of 29 kD, 28 kD, 65–67 kD, 15 kD, and about 200 kD for CR, CB, GAD, PV, and SV2 respectively.
Western blots were used to examine antibody specificity (). A gecko brain was removed after the animal had been deeply anesthetized as described above. Pre-chilled RIPA buffer containing a protease inhibitor mixture was added at a volume of 5 ml/g tissue, and the tissue was broken down by homogenizer and vortex for 60 seconds. The suspension was stored on ice for 45 minutes, followed by centrifugation at 14,000g for 10 minutes at 4°C. Cytosolic protein was collected from the supernatant, whose concentration was measured by Bradford protein assay (Bio-Rad, Hercules CA). Ten micrograms protein and 5 μl standard (161-0324; Bio-Rad) were loaded onto 12% SDS-PAGE gels. The protein was separated by SDS-PAGE and transferred to a PVDF membrane. The membrane, blocked with TBST containing 3% BSA for 1 hour at room temperature, was incubated in primary antisera diluted to 1:2,000 in 1% BSA in TBST overnight at 4°C. Afterward, it was probed with alkaline phosphataseconjugated goat anti-rabbit or goat anti-mouse (1:5,000; 31340 and 31320; Pierce, Rockford, IL) for 1 hour at room temperature. The blots were visualized by exposing the membrane to ECF substrate (Amersham Bioscience, Piscataway, NJ) in the dark for 5–10 minutes and scanned on Storm (Molecular Dynamics, Sunnyvale, CA).
Blots with CR showed a major band at an estimated molecular weight of 29 kD, similar to the measurement in chick (Hack et al., 2000
). CB, PV, and GAD blots also yielded single bands at estimated molecular weights of 28 kD (Ellis et al., 1991
), 15 kD (Celio, 1990
; Lohmann and Friauf, 1996
) and 65–67 kD (Sloviter and Nilaver, 1987
), respectively (). Western blot analysis of the anti-SV2 antibody had previously been tested against zebra finch brain tissue and produced a broad band over a range of protein sizes of 66–200 kD (Nealen, 2005
). This may be due to the difficulty in electrophoretically resolving glycoproteins. Our Western blot of SV2 against gecko brain tissue showed multiple bands around 200 kD, within the range of results for zebra finch ().