Production and characterization of antibodies against human AHI1 and mouse Ahi1
Rabbit polyclonal antibodies specific for human AHI1 or mouse Ahi1 were raised against various peptides derived from the human or mouse protein sequence, respectively. Specifically, peptides generated for the human AHI1 protein (Ab-813; PAPQKQSINKNKSQ, amino acids 1138-1151) and to four distance separate amino acid locations in the mouse Ahi1 protein (N-terminus: Ab-82/83, PTADDSDDSREKTGIE, amino acids 21-36; C-terminus: Ab-809, RSPPLTPKEKTKPE, amino acids 973-986; C-terminus: Ab-84/85, SEKGKDQNVEDRGHK, amino acids 1014-1028; C-terminus: Ab-05/06, KKSEPVVRKVTLIE, amino acids 1034-1047). The synthesis and conjugation of the peptide and production of rabbit polyclonal antibodies were performed by either Covance (Denver, PA) or Sigma-Genosys (The Woodlands, TX). All antibodies were affinity purified using the corresponding peptide and the SulfoLink Kit (Pierce, Rockford, IL) according to the manufacturer’s instructions.
The specificity of each antibody was determined using standard Western blotting and immunostaining techniques. Full-length human AHI1 and mouse Ahi1 sequences were cloned in-frame into an EGFP expression plasmid containing a CMV promoter (Human: N-terminal EGFP-AHI1 and C-terminal EGFP-AHI1; Mouse: N-terminal EGFP-Ahi1 and C-terminal EGFP-Ahi1). HEK293 cells (ATCC, Manassas, VA) were transiently transfected with each of these constructs using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After EGFP-expressing cells were observed (approximately 70% of the cells at confluence in each culture dish expressed EGFP), they were lysed with boiling 2X sample buffer (62.5 mM Tris (pH 6.8), 2% SDS, 20% glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol (BME)), yielding protein lysates for separation on SDS/8% PAGE gels with electroblotting onto PVDF transfer membrane (Millipore, Billerica, MA). Blots were probed with each AHI1/Ahi1 antibodies (rabbit IgG) in combination with EGFP antibodies in order to determine localization of both AHI1 and EGFP; co-immunoreactivity indicates the specificity of each of the AHI1/Ahi1 antibodies (EGFP antibody: immunogen: full-length Aequorea victoria green fluorescent protein; mouse IgG; 1:2000; Cat. # 632381; Lot # 8010026; Clontech, Mountain View, CA); this antibody recognizes native and denatured GFP and EGFP as well as N- and C-terminal fusion proteins; addition of this antibody to non-EGFP containing cultured neuronal cells or brain tissue results in no specific signal (data not shown; see manufacturer’s technical data sheet). The N-terminal EGFP-human-AHI1 and C-terminal EGFP-human-AHI1 lysates were probed with Ab-813; and the N-terminal EGFP-mouse-Ahi1 and C-terminal EGFP-mouse-Ahi1 lysates were probed with Ab-82/83, Ab-809, Ab-84/85, and Ab-05/06 at varying concentrations. The primary antibodies were detected with goat anti-mouse IgG-Cy3 (for EGFP) and goat anti-rabbit IgG-Cy5 (for AHI1) fluorescent secondary antibodies (1:500; GE Healthcare, Piscataway, NJ). The signals were analyzed with a Typhoon Trio+ scanner and ImageQuant analysis software (GE Healthcare). Western blot analysis of these lysates demonstrated that the human AHI1 antibody recognized a band of ~190 kDa corresponding to the size of the EGFP-human-AHI1 fusion protein. Similar results were obtained for the mouse Ahi1 antibodies and the EGFP-mouse-Ahi1 fusion protein (~160 kDa). The EGFP antibody detected an overlapping molecular mass signal with the AHI1/Ahi1 antibody signals (data not shown). In addition, incubation of each of the AHI1/Ahi1 antibodies with a 5X excess of the corresponding peptide was able to completely abolish the signal corresponding to the AHI1/Ahi1 bands (data not shown).
In addition, HEK293 cells that expressed EGFP-tagged human AHI1 or mouse Ahi1 were also immunostained with the corresponding species AHI1/Ahi1 antibodies in conjunction with an Alexa-546 goat anti-rabbit IgG secondary antibody (Invitrogen), to determine whether AHI1/Ahi1 and EGFP co-localized in these cells. Over-expression of human AHI1-EGFP and mouse Ahi1-EGFP by transient transfections resulted in cytoplasmic expression of the EGFP-tagged AHI1/Ahi1 proteins. In these transfected cell lines, immunostaining with the corresponding human AHI1 antibody or mouse Ahi1 antibodies demonstrated overlapping localization of EGFP and human AHI1 and mouse Ahi1, respectively (data not shown).
To further confirm the specificity of the mouse Ahi1 antibodies, we performed peptide blocking experiments in which the mouse Ahi1 antibodies were pre-incubated with a 5X excess of each corresponding peptide that was used to raise the antibodies. The antibody-peptide mixture was then added to mouse brain sections using standard immunohistochemistry (as described below). Excess peptide was able to completely abolish the immunostaining observed with the mouse Ahi1 antibodies alone (without peptide) ().
Figure 1 Characterization of the specificity of Ahi1 antibodies. A, Immunohistochemical detection of Ahi1 shows diffuse cytoplasmic staining in the mature murine hypothalamus. Bar indicates 200 μm. B, Lack of detection of Ahi1 immunoreactivity in the presence (more ...)
An additional confirmation method used to determine the specificity of each mouse Ahi1 antibody was Western blot analyses of IMCD3 cells (a mouse inner medullary collecting duct cell line; ATCC, Manassas, VA). IMCD3 cells were grown according to the manufacturer’s instructions. IMCD3 cells were transfected with a pSUPER.gfp+neo RNAi construct (Oligoengine, Seattle, WA) containing a shRNAi sequence directed against mouse Ahi1 or a scrambled sequence. After 3-5 days in culture, the IMCD3 cells were lysed and processed for mouse Ahi1 Western immunoblotting as described above. Wild-type IMCD3 cells or cells transfected with scrambled sequence expressed full-length Ahi1 with a molecular mass of 130 kDa as determined by Western blotting with Ahi1 antibodies (). One additional ~150 kDa protein band was also observed by Western blotting in wild-type IMCD3 cells and cells transfected with scrambled sequence (data not shown). No bands were found with a molecular mass below 130 kDa. IMCD3 cells transfected with shRNAi constructs against mouse Ahi1 demonstrated no detectable levels of the 130 kDa, full-length mouse Ahi1 protein (). However, the additional ~150 kDa protein band still remained in Ahi1-knockdown cells (data not shown). Given this result, we performed immunocytochemistry for Ahi1 in Ahi1-knockdown IMCD3 cells. Significant decreases in Ahi1 immunoreactivity were observed in these cells (data not shown). This suggests that this ~150 kDa band observed by Western blotting was not observed in non-denatured, immunocytochemical experiments.
The final confirmation method used to determine the specificity of each mouse Ahi1 antibody was immunostaining of primary mouse neurons that had been transfected with mouse Ahi1
siRNAs (details below). Primary neuronal cultures from mouse hypothalamus were grown using modified neuronal culturing techniques (Banker and Goslin, 2002
). Briefly, hypothalami from embryonic day 17.5 (E17.5) mice were dissected and placed into ice-cold Ca++
free PBS. The tissue was minced into smaller pieces and added to a trypsin/EDTA solution for 30 minutes at 37°C. Following trypsin digestion, the tissue was rinsed with a trypsin inhibitor and placed in Neurobasal medium supplemented with B27, glucose, sodium pyruvate, glutamine, and gentamicin (all at manufacturer’s suggested concentrations (Invitrogen (GIBCO), Carlsbad, CA)). The tissue was triturated multiple times, to liberate individual cells from the tissue. Viable cells were counted on a hemocytometer, using the trypan blue exclusion assay, and were then plated onto poly-d-lysine treated glass coverslips at density of 2000-5000 cells/mm2
. Following incubation for 1-3 days, hypothalamic cell cultures were transfected, using Lipofectamine 2000 (Invitrogen), with both an ON-TARGETplus SMARTpool of siRNAs directed against mouse Ahi1
(Dharmacon, Chicago, IL) and an EGFP-expressing plasmid, all according to the manufacturers’ suggested protocols. Neuronal cultures were allowed to grow for an additional 3-5 days and were then fixed in cold 4% paraformaldehyde (PFA) and processed for mouse Ahi1 immunostaining, as described below. The transfected cells were visualized on a Zeiss AxioImager-A1 microscope with AxioVision Rel. 4.5 software (Carl Zeiss Microimaging, Thornwood, NY), with EGFP used as the indicator of any cell that was co-transfected with mouse Ahi1
Primary cultures of hypothalamic neurons were immunostained with each mouse Ahi1 antibody individually; they demonstrated diffuse cytoplasmic, dendritic, and axonal localization (). However, knockdown of mouse Ahi1 expression in hypothalamic cultures by siRNA resulted in greatly reduced expression or absence of Ahi1 staining only with mouse Ahi1 antibodies, Ab-84/85 and Ab-05/06 (data for Ab-05/06 shown in ). Antibody Ab-82/83 continued to show immunostaining in cultured Ahi1-knockdown neurons, although at a reduced level, suggesting that this antibody was not entirely specific for mouse Ahi1 in non-denatured protein preparations (this antibody is specific for mouse Ahi1 protein by Western blotting). Lastly, Ab-809 did not show any detectable immunostaining of cultured neurons suggesting the epitope for this antibody is hidden in the folded protein, under non-denaturing conditions (since this antibody is specific by Western blotting). Given that all of the mouse Ahi1 antibodies are specific by Western blotting, but only mouse Ab-84/85 and mouse Ab-05/06 are specific in immunohistochemical assays, we present all of following mouse experiments using Ab-05/06; however, Ab-84/85 gave similar results by immunohistochemistry and Western blotting. For experiments using human AHI1 protein, we utilized human AHI1 antibody, Ab-813.
Animals and histological procedures
All mice used to characterize the normal expression pattern of Ahi1 were wild-type Swiss Webster mice obtained from Taconic (Germantown, NY). The mice were sacrificed at various developmental ages: embryonic day (E) 10.5, E12.5, E14.5, E16.5, E18.5, postnatal day (P) 0.5, P1.5, P3.5, P7.5, P10.5, P14.5, P21.5, and adult.
To obtain tissue for immunohistochemistry (IHC), we administered an overdose of either sodium pentobarbital or avertin to mice, which were then perfused transcardially with 0.1M phosphate-buffered saline (PBS; pH 7.4). This was followed by cold 4% PFA (made in PBS). Brains were dissected, post-fixed in 4% PFA, and stored at 4°C for up to 7 days. Following post-fixation, brains were cryoprotected in a 30% sucrose solution made in PBS.
Brains were sectioned on a Microm cryostat (Richard-Allan Scientific, Kalamazoo, MI), in either the sagittal or coronal plane, at a thickness of 25-40 μm (depending on the developmental stage). All sections were mounted on Superfrost Plus microscope slides (Fisher Scientific, Pittsburg, PA) and allowed to air dry for 30 minutes before being stored at -80°C.
All mouse procedures were performed under approval from the Institutional Animal Care and Use Committees of both Rensselaer Polytechnic Institute and the Wadsworth Center (NY State Department of Health), in accordance with The National Institutes of Health Guide for the Care and Use of Laboratory Animals.
AB and TL Zebrafish were maintained under a 10 hour dark to 14 hour light cycle at 28.5°C using standard laboratory practices (Nüsslein-Volhard and Dahm, 2002
). Zebrafish embryos were obtained by natural mating and were staged by days post-fertilization (dpf) and according to standard morphological criteria (Kimmel et al., 1995
). Embryos were raised at 28.5°C until sacrificed and fixed at the desired stage. 1-phenyl-2-thiourea (0.003%) was used to suppress pigmentation of embryos older than 24 hours post-fertilization (hpf). All fish procedures were performed under approval from the Institutional Animal Care and Use Committee of Rensselaer Polytechnic Institute.
Tissue was obtained for Western blotting from additional mice at the ages described previously. Mice were administered an overdose of either sodium pentobarbital or avertin, and their brains and peripheral organs were harvested and immediately frozen in liquid nitrogen. For some animals, brains were dissected into sub-regions (cerebral cortex, hippocampus, striatum, thalamus, hypothalamus, brainstem, and cerebellum) and frozen in liquid nitrogen. All samples were stored at -80°C until processed into lysates.
Whole mouse brain (or sub-regions) or peripheral tissues were homogenized in RIPA buffer (50 mM Tris (pH 8), 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride and 1X protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN)) with a plastic mortar and pestle. Homogenized lysates were incubated on ice for 30 minutes and centrifuged at 10,000 x g for 30 minutes to obtain the supernatant. The protein concentration was determined for each supernatant sample using the Advanced Protein Assay reagent (Cytoskeleton Inc., Denver, CO) according to the manufacturer’s protocol. Protein lysates (5 μg of protein), isolated from brains of variously aged mice, from various regions of mouse brain, or from various peripheral organs, were resolved by SDS/8% PAGE gel electrophoresis and electroblotted onto PVDF transfer membrane. The membrane was blocked with 3% non-fat dried milk in TBS-TX (100 mM Tris (pH 7.4), 150 mM NaCl, 0.01% Triton X-100) for 1 hour at room temperature and then probed with the various mouse Ahi1 antibodies (at 1:1000 to 1:4000 dilution in blocking solution) and with anti-βIII tubulin antibodies (chicken IgY; 1:4000; Chemicon, Temecula, CA) as a loading control, at room temperature overnight.
For human brain tissue Western blotting, a custom human normal brain tissue blot was obtained (ProSci, Poway, CA) containing the following tissues: adult human amygdala, cerebellar peduncles, cerebellar hemispheres, cerebellar vermis, cerebral cortex, and medulla oblongata; and fetal human cerebellum and brainstem. Briefly, 15 μg of each protein lysate were separated on a 4-20% gradient SDS-PAGE gel and transferred onto nitrocellulose membranes. The membrane was blocked with 3% non-fat dried milk in TBS-TX for 1 hour at room temperature and then probed with our human AHI1 antibody (Ab-813; 1:100) and with anti-βIII tubulin antibodies (chicken IgY; 1:10000; Chemicon) as a loading control, at room temperature overnight.
Primary antibodies were detected with the SuperSignal West Femto Maximum Sensitivity Substrate Chemiluminescence Kit (Pierce, Rockford, IL). The signal was analyzed with a Typhoon Trio+ scanner and ImageQuant analysis software (GE Healthcare).
All tissue sections were allowed to dry at room temperature before being permeabilized for 10 minutes in 0.04% Triton X-100 in PBS (pH 7.4; PBS-TX). Endogenous peroxidases were removed via incubation in 0.03% hydrogen peroxide/methanol for 30 minutes. This hydrogen peroxide step was excluded if antibody detection was accomplished through fluorescently tagged antibodies. Sections were then washed in PBS-TX and blocked for 1 hour in 10% normal goat serum (NGS), followed by an overnight incubation with the primary antibody. For the majority of the immunohistochemistry, we utilized the mouse Ahi1 antibody designated Ab-06 (as described above) at a concentration of 1:1000 in 1% NGS/PBS-TX. However, the other Ahi1 antibodies, Ab-05 and Ab-84/85, gave similar results. Tissue sections were again washed in PBS-TX, followed by primary antibody detection using the Vector Elite ABC kit and a 3,3′-diaminobenzidine (DAB) kit (Vector Labs, Burlingame, CA) according to the manufacturer’s instructions. The slides were allowed to dry overnight before a coverslip was applied with Cytoseal-60 (Richard-Allan Scientific, Kalamazoo, MI); slides were visualized with a Zeiss AxioImager-Z1 microscope and imaged with an AxioCam MRc camera with AxioVision Rel. 4.5 software and MosaiX (Carl Zeiss Microimaging). All images were processed using Adobe Photoshop CS2 (version 9.0.2; Adobe Systems Inc., San Jose, CA). Contrast and brightness of images were adjusted through linear level adjustments, as needed, to optimize the clarity of the images presented.
For fluorescence immunohistochemistry, we used the following antibodies: NeuN (immunogen: purified cell nuclei from mouse brain; clone: A60, mouse IgG; Cat. # MAB377; Lot # 060109159; Chemicon, Temecula, CA); this antibody recognizes the neuron-specific protein NeuN present in most neurons of the vertebrate CNS (Bulloch et al., 2008
) and shows immunoreactivity in morphologically distinct neurons in culture (data not shown) and in brain tissue (Ferland et al., 2003
); glial fibrillary acidic protein (GFAP)(immunogen: purified bovine GFAP; chicken IgY; Cat. # AB5541; Lot # 0605030036; Chemicon); this antibody recognizes GFAP in most astrocytes of the vertebrate CNS, reacts with both native and recombinant protein, and it shows immunoreactivity in morphologically distinct glia in culture (data not shown; see manufacturer’s technical data sheet); LR11 (immunogen: human LR11 a.a. 1220-1337; clone: 48, mouse IgG; Cat. # 611860; Lot # 74851; BD Transduction Laboratories, San Jose, CA); this antibody recognizes the lipoprotein receptor homologue that is expressed at high levels in the brain (Posse De Chaves et al., 2000
) and it recognizes a 250 kDa band corresponding to the predicted mass of LR11. Increased dilutions of the primary antibody result in decreasing immunoreactivity (see manufacturer’s technical data sheet). LR11 antibodies also detect morphologically distinct organelles known as stigmoid bodies (Gutekunst et al., 2003
), therefore making LR11 an ideal marker for the stigmoid body; dynein (immunogen: bovine brain cytoplasmic dynein (the antigenic site has been localized to the first 60 a.a. of the N-terminus of dynein); clone: 74.1, mouse IgG; Cat. # MAB1618; Lot # 0512018659; Chemicon); this antibody recognizes the 74 kDa intermediate chain subunit of cytoplasmic dynein (see manufacturer’s technical data sheet). Dynein antibodies demonstrate immunoreactivity to neuronal growth cones (Grabham et al., 2007
). The final working concentrations for the primary antibodies were as follows: Ahi1 (1:1000), NeuN (1:1000), GFAP (1:200), LR11 (1:1000), and dynein (1:100). Omission of all primary antibodies, used in the present studies, resulted in the absence of any immunoreactivity (data not shown).
Tissue sections or cells on slides were washed extensively in 0.1M PBS (pH 7.4). Then, they were washed in PBS-TX followed by blocking in 10% NGS made in PBS-TX, for at least 1 hour. Primary antibodies were diluted to their final working concentrations in PBS-TX containing 1% NGS. The slides were incubated overnight at 4°C. The following day, all slides were washed in PBS-TX and then were incubated for at least 2 hours with the appropriate fluorophore-labeled secondary antibodies: Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 546 goat anti-rabbit IgG, and Alexa Fluor 488 goat anti-chicken IgY (all from Invitrogen (Molecular Probes), Carlsbad, CA). Each fluorophore-labeled secondary antibody was diluted 1:500 in PBS-TX containing 1% NGS. Following the 2 hour incubation with the fluorophore-labeled secondary antibodies, all sections were rinsed in PBS and mounted on slides, and a coverslip was applied with Fluoromount-G (Southern Biotechnology, Birmingham, AL). All images were visualized with a Zeiss AxioImager-Z1 microscope and imaged with an AxioCam MRm camera and AxioVision Rel. 4.5 software. All images were processed using Adobe Photoshop CS2. Contrast and brightness of images were adjusted through linear level adjustments, as needed, to optimize the intensity range of the images.
In situ hybridization probes for zebrafish ahi1
Total RNA was isolated from zebrafish embryos at various stages using TRIzol reagent according to the manufacturer’s instructions. PCR and genomic analysis indicated that ahi1 is a single gene in zebrafish. Gene-specific PCR primers were designed according to Ensembl (Ensembl release 47, Assembly Zv7) for the zebrafish ahi1 transcript (Ensembl ID: ENSDARG00000044056). Zebrafish ahi1 cDNA was obtained by reverse transcription, 3′-RACE, and 5′-RACE, following manufacturer’s instructions (GeneRace Kit, Invitrogen, Carlsbad, CA). PCR reactions were carried out using primers with T3/T7 polymerase binding sites, and with zebrafish ahi1 cDNA as template. Digoxigenin-11-uridine-5′-triphosphate (DIG)/NTP mix (10 mM ATP, 10 mM GTP, 10 mM CTP, 6.5 mM UTP, 3.5 mM DIG-11-UTP in Tris-neutralized solution, pH 7.5)(Roche, Indianapolis, IN) was used instead of dNTP. PCR products with the correct sizes were transcribed with T3/T7 polymerase following the manufacturer’s instructions (MEGAscript T3 Kit, MEGAscript T7 Kit, Roche). Primer pairs used to generate the five riboprobes used in this study are available upon request. The probes were, in order from the 5′- to the 3′-end of the zebrafish cDNA: probe 1: nt 15-468; probe 2: nt 15-589; probe 3: nt 15-661; probe 4: nt 389-661; probe 5: nt 15-389. Staining patterns for the individual probes were indistinguishable.
Whole-mount in situ hybridization
Zebrafish embryos from 48 hpf to 5 dpf were sacrificed and fixed overnight in 4% PFA/PBT (0.1% Tween-20 in PBS) at 4°C, and were then transferred to methanol for at least 16 hours at -20°C. Embryos were washed in 75%-, 50%-, 25%-methanol/PBT gradients, followed by washes in PBT. Embryos at 2 dpf, 3 dpf, 4 dpf, 5 dpf were treated with 10 μg/ml proteinase K for 20 , 22 , 24 , 26 minutes, respectively, followed by a 30 minute incubation in 4% PFA/PBT and washes in PBT at room temperature. Embryos were prehybridized in HYB buffer (50% formamide, 5X SSC, 500 μg/ml torula RNA, 50 μg/ml heparin, 0.1% Tween-20, 9 mM citric acid at pH 6.0-6.5) for 2 hours at 68°C and then hybridized with ~150 ng of probe in HYB buffer overnight at 68°C.
On the next day, embryos were washed sequentially with 75% HYB/2X SSC, 50% HYB/2X SSC, 25% HYB/2X SSC, 2X SSC, 10 minutes for each step, followed by two 30 minute washes with 0.2X SSC at 68°C. Embryos were then equilibrated to room temperature and washed sequentially with 75% 0.2X SSC/PBT, 50% 0.2X SSC/PBT, 25% 0.2X SSC/PBT, and then PBT, 5 minutes for each wash. Embryos were incubated in 0.1 M glycine (pH 2.2) + 0.1% Tween-20 at room temperature, to remove endogenous alkaline phosphatases; this was followed by washes in PBT. Blocking was performed in PBT with 2 mg/ml BSA and 5% normal calf serum for 2 hours at room temperature. After blocking, embryos were incubated in PBT/BSA with anti-DIG antibody (Anti-digoxigenin, Fab fragments; Roche) at 1:2000 overnight at 4°C.
Embryos were washed in PBT/BSA and then further washed in NTMT buffer (0.1 M Tris-Cl (pH 9.5), 0.1 M NaCl, 0.05 M MgCl2, 0.1% Tween-20) at room temperature. Staining was then carried out in the dark at room temperature with 225 μg/ml NBT (4-Nitro blue tetrazolium chloride; Roche) and 175 μg/ml BCIP (5-bromo-4-chloro-3-indolyl-phosphate; Roche) in NTMT, until staining became visible. Embryos were imaged on a Zeiss AxioImager-Z1 microscope with a Zeiss AxioCam MRc camera. Figures were prepared using AxioVision 4.5 software and Adobe Illustrator; image manipulations were limited to linear level adjustments, rotations and scaling.
The hypothalamus was dissected from E18.5 mouse brains and then fixed in either of two electron microscopy-compatible fixatives. The hypothalamus was processed either for immuno-electron microscopy or for preservation of fine structure.
For immuno-electron microscopy, the tissue was fixed with cold 4% PFA/0.1% glutaraldehyde in PBS (pH 7.4) for 2 hours, washed twice in water, dehydrated in a graded ethanol series, and embedded in LR White. Semi-thin (0.08-0.20 μm) sections were cut using a Diatome diamond knife on a Reichert Ultracut E ultramicrotome and were placed on carbon-coated, Formvar-coated hexagonal nickel grids. The sections were blocked for 2 hours in a blocking solution containing 40 μg/ml goat sera, 20 μg/ml BSA in TBS buffer (containing 1 μg/ml BSA, 0.05% Tween-20, 0.5 M NaCl, 20 mM NaN3, pH 7.4), exposed to the anti-Ahi1 rabbit polyclonal antibody (Ab-05) in TBS buffer (4.0 μg/ml, pH 7.4). Following an overnight incubation in the Ahi1 antibody, the sections were washed four times in TBS, and then labeled with a 1:100 dilution of 10-nm gold particles conjugated to goat anti-rabbit IgG antibodies (Ted Pella Inc., Redding, CA) in TBS buffer with 10 μg/ml goat sera for 2 hours. Sections then were washed five times in TBS, and the antibodies were covalently linked with 1% glutaraldehyde in water. The samples were stained with uranyl acetate for 15 minutes, followed by three water washes. Substitution of rabbit IgG for the primary antibody provided a negative control. Sections were analyzed at 80 kV with a Zeiss 910 transmission electron microscope (Carl Zeiss, Oberkochen, Germany).
For fine structure studies, the tissue was fixed in cold 6.5% glutaraldehyde in PBS (pH 7.4) for 2 hours, washed two times, 10 minutes per wash in PBS, and rinsed in 0.1 M sodium cacodylate buffer (pH 7.4) twice, 10 minutes per rinse. Samples were post-fixed in 1% osmium tetraoxide in cacodylate buffer for 1 hour, washed in water overnight, dehydrated in a graded acetone series, and embedded in Epon 812/Araldite. Semi-thin (0.08-0.20 μm) sections were stained with uranyl acetate and Reynold’s lead. Sections were viewed on a Zeiss 910 transmission electron microscope at 80 kV.