Conjugation and purification of GTTR
An excess of gentamicin (in K2
, pH 10) was mixed with Texas Red (TR) succinimidyl esters (Invitrogen, CA) to minimize the possibility of over-labeling individual gentamicin (GT) molecules with more than one TR molecule and to ensure the polycationic nature of the conjugate (GTTR), as previously described (Sandoval et al. 1998
). After conjugation, the reaction mixture was separated by reversed phase chromatography using C-18 columns (Burdick and Jackson, Muskegon, MI) to purify the conjugate from unconjugated gentamicin and potential contamination by unreacted TR (Myrdal et al. 2005
). The isolated GTTR conjugate was aliquoted, lyophilized, and stored desiccated, in the dark at -20°C until required.
Wild-type and mariner
zebrafish larvae, 5 days after fertilization, were treated with a dose range of gentamicin, GTTR, or unconjugated Texas Red (up to 100 mg/ml in standard E3 medium (Mullins et al. 1994
; Westerfield 1993
) for 1 h and allowed to recover for 4 h prior to fixation in 4% formaldehyde containing 0.5% Triton X-100 (Myrdal et al. 2005
). Larvae were then labeled with Alexa-488-conjugated phalloidin to determine the number of surviving hair cell bundles by confocal microscopy, similar to that described previously by Harris et al (2003
). For time course studies, wild-type and mariner
zebrafish larvae were treated with 1.6 mg/ml GTTR or unconjugated Texas Red for up to 20 min prior to washing, fixation, and phalloidin labeling as described above. For competition studies, wild-type zebrafish larvae were treated with 1.6 mg/ml GTTR with or without 10 mM Ca++
or 1.6 mg GT for 10 min, prior to fixation and phalloidin labeling.
Mice (C57/BL6; 21–28 days old) received one intra-peritoneal (i.p.) injection of 2 mg/kg GTTR (in phosphate-buffered saline (PBS), pH 7.4) and at various time points (10, 20, and 30 min and 3, 6, 9, and 24 h) were anesthetized and serum collected prior to cardiac perfusion with PBS, then 4% formaldehyde. Cochleae and kidneys were excised and post-fixed in 4% formaldehyde containing 0.5% Triton X-100 for 45 min, washed, labeled with Alexa-488-conjugated phalloidin, rinsed, and post-fixed with 4% formaldehyde (Myrdal et al. 2005
). Alternatively mice received 0.2, 2, 20, or 200 mg GT, prior to the above procedures. Serum levels of GTTR and gentamicin were determined using standard diagnostic particle-enhanced turbidimetric inhibition immunoassay methods (Newman et al. 1992
) on a Beckman Synchron Drug Calibrator 3 Plus, with a sensitivity of 0.5 mg/ml.
For cryostat sections, cochleae were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) containing 2% formaldehyde for 1 week at 4°C (EDTA plus fixative solution replaced daily), infiltrated with OCT, cryostat sectioned at 8 mm, and mounted on gelatin-coated glass slides. For high resolution confocal microscopy of wholemounted tissues, cochleae were rinsed, the bone covering the basal coil of the cochlea removed and the lateral wall (spiral ligament and stria vascularis) excised. Kidneys were vibrotome-sectioned at 100 mm. All GTTR-treated specimens were then labeled with Alexa-488-conjugated phalloidin to localize filamentous actin. In some cases, the fixed and excised lateral wall was cut into thin slices using a fresh scalpel blade for each cut and positioned between thin agar strips, with the cut surfaces parallel to the slide, to present a “cross-sectioned” lateral wall for image acquisition as high resolution xy optical sections.
Immunocytochemistry For immunolocalization of GT, specimens were immunoblocked in 10% goat serum in PBS for 30 min and then incubated with 50 mg/ml rabbit polyclonal anti-GT antisera (American Qualex, San Clemente, CA) for 1 h. After washing with 1% goat serum in PBS, specimens were further incubated with 20 mg/ml Alexa-488-conjugated goat-anti-rabbit antisera (Molecular Probes, Eugene, OR) for 45 min, washed, counter-labeled with Alexa-568-conjugated phalloidin, post-fixed with 4% formaldehyde for 15 min, and washed again. For immunocytochemical controls, primary antiserum was omitted or replaced with GT-adsorbed antiserum and immunoprocessed as above.
Cell culture Madin-Darby canine kidney distal tubule (MDCK) cells were cultured in antibiotic and phenol red-free Dulbecco’s minimal essential medium (MEMa, Invitrogen, Ca) with 10% fetal bovine serum at 37°C with 5% CO2, 95% air. For experiments, cells were seeded into eight-well coverglass chambers (ISC BioExpress) at 3,000 cells/well and grown for 5 days, when they had become confluent, cuboidal, and had time to develop tight junctions. Cells were washed three times with phosphate-buffered saline (containing 1.25 mM calcium) and treated with a dose range of GTTR for 30 s at 20°C (precluding endocytosis). Following treatment, cells were rinsed three times with buffer then fixed and delipidated with 4% formaldehyde plus 0.5% Triton X-100 (FATX) for 45 min. Following fixation, cells were thoroughly rinsed with phosphate buffered saline (Invitrogen, CA) prior to imaging. Live cells were also incubated with a dose range of GT for 30 s, prior to rinsing, fixation as above, and then subsequent immunolabeling procedures as described above.
All specimens were mounted on slides, immersed in VectaShield (Vector Labs, CA), coverslipped and examined using a Bio-Rad MRC 1024 ES laser scanning confocal system attached to a Nikon Eclipse TE300 inverted microscope. Alexa-488 and Texas Red images were collected sequentially using 1,024
1,024-pixel box size using a 60× lens (n.a. 1.4) with an xy
230 nm and xz
440 nm (Steyger et al., 2003
). All specimens were imaged at the same laser intensity and gain settings, including control tissues. Each time point or dose group (n
3) was conducted simultaneously with the standard time point (30 min) group, against which ratiometric image analysis was conducted (see below). Representative images from each experiment were identically prepared for publication using Adobe Photoshop.
Image analysis For image analysis, optical sections from each experimental set were identified and regions of interest (marginal cells [minus nuclei], intra-strial tissues [putatively intermediate cells], basal cells, and fibrocytes) were manually segmented for pixel intensity determination (ImageJ, NIH). To normalize data between experimental sets, the mean intensity was ratioed against the standard (intensity of marginal cells 30 min after GTTR only injection) and plotted. Student’s t test was used to determine any significant difference between time points, dosing groups or for regions of interest in the same specimens.