Leica tcs spe confocal microscope

Frozen sections were rinsed with PBS and incubated in blocking solution (10% goat serum in PBS with 0.2% Triton X-100) for 60 minutes at room temperature. The samples were then incubated overnight at 4°C with the following primary antibodies: rabbit monoclonal anti-myosin7a (Abcam, ab155984) and rabbit monoclonal anti-neurofilament heavy polypeptide (NF200, Abcam, ab40796) and then incubated with Alexa-488-conjugated goat anti-rabbit secondary antibodies (Yeasen, 33106ES60) for 1 hour at room temperature. F-actin was stained using Alexa-568-labeled phalloidin (1:100; Invitrogen) for 1 hour at room temperature. Nuclear staining was performed with 2 mg/mL DAPI (Invitrogen). The fluorescence images were captured using a Leica TCS-SPE confocal microscope (Leica Microsystems).

The CDDP-induced hearing loss model was obtained as previously described.23 (link) The guinea pigs were randomly assigned into 5 groups, with n=10 mice/group. 1) CDDP, 2) CDDP + A666, 3) CDDP + DEX, 4) CDDP + DEX-NP, and 5) CDDP + A666-DEX-NP. Briefly, a dose of 12 mg/kg of CDDP was administered by intraperitoneal injection. DEX (120 µg/mL, 5 µL), DEX-NP (120 µg/mL, 5 µL), and A666-DEX-NP (120 µg/mL, 5 µL) were administered onto the RWM 1 hour before CDDP injection.
The auditory function was measured by auditory brainstem response (ABR) recording. The ABR thresholds were measured at frequencies of 4, 8, 16, and 24 kHz 1 day before drug administration and 3 days after, as previously reported.23 (link)
After the 3-day ABR measurements postdrug administration, the animals were anesthetized with lethal doses of 1% pentobarbital sodium and subjected to intracochlear perfusion with 4% PFA in PBS. The temporal bones were decalcified in 0.12 M EDTA in PBS. The hair cell damage was evaluated in whole mounts from groups of guinea pigs treated with different formulation groups. Myosin 7a (1:200) was used to identify cochlear hair cells. These whole mounts were examined using a Leica TCS SPE confocal microscope (Leica Microsystems, Wetzlar, Germany). The number of remaining OHCs was counted in a 1 mm long strip at the region, approximately 7–10 mm from the apex.

Cardiomyocytes were seeded on 35 mm glass-bottom culture dishes (Matsunami Glass, Bellingham, WA) coated with fibronectin and gelatin, and were cultured and transduced as described above. The cells were fixed in 4% paraformaldehyde (Millipore-Sigma, Burlington, MA) in PBS for 20 min and permeabilized with saponin (Millipore-Sigma) in PBS for 1 h at room temperature in preparation for staining of nuclear antigens. Samples were washed three times (5 min each time) with PBS under light rotation and blocked with 5% BSA in PBS for 1 h. Antibodies against Connexin-43 (rabbit; Abcam, cat. no. ab11370, Cambridge, MA), α-Actinin (mouse, Sigma, cat. no. A7811, St. Louis, MO), or mCherry (goat, OriGene, cat. no. TA150126, Rockville, MD) were added overnight at 4 °C in 1% BSA in PBS. After three washes with PBS, cells were then incubated with a corresponding secondary antibody conjugated at room temperature for 1 h (anti-rabbit Alexa Fluor 488, anti-mouse Alexa Fluor 647, anti-goat Cy^™3, Jackson ImmunoResearch Inc., West Grove, PA) in 1% BSA in PBS. After 3 washes with PBS, nuclear DNA was stained with DAPI (Millipore-Sigma, Waltham, MA) for 10 minutes. Following another three washes in PBS, VECTASHIELD^® Antifade Mounting Medium (Vectorlab, Newark, CA) was added to the coverslip. Samples were visualized with a Leica TCS SPE confocal microscope (Leica Microsystems, Wetzlar, Germany).