Fluorescein isothiocyanate conjugated phalloidin

For the IF studies, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline. Fixed cells were incubated with 1:2000 fluorescein isothiocyanate-conjugated phalloidin (Sigma, St. Louis, MO) or antibodies as indicated. Cells were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (Calbiochem, San Diego, CA) and imaged with a confocal laser-scanning microscope (Olympus FV1000, Tokyo, Japan).

To evaluate any morphological changes induced by the treatments, phalloidin staining was performed for each experimental group. For this purpose, ARPE-19 cells were seeded at 2 × 10⁵ cells/well density in 6-well plates. Cells were washed two times with Phosphate-Buffered Saline (PBS), were fixed with 4% Paraformaldehyde (PFA) for 10 min, and were incubated with 3% Bovine Serum Albumin (BSA) with 0.1% Triton for 30 min for non-specific binding sites block. Fluorescein Isothiocyanate conjugated Phalloidin (Sigma Aldrich, Saint Louis, MO, USA) (1:250 in PBS1X), incubated for 40 min at Room Temperature (RT), was used to label cellular cytoskeleton and Bisbenzimide to counterstain cellular nuclei. Images were acquired with Floid Cell Imaging Station (Thermo Fisher Scientific Inc., Monza, Italy).

Proteins were separated on SDS–PAGE and transferred to nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5 % non-fat milk and incubated with the corresponding mouse anti-FOS, Met, E-cadherin, α-catenin, β-catenin, N-cadherin, fibronectin, vimentin (BD Biosciences, 1:1000 dilution), α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, 1:1000 dilution), snail and GAPDH (Cell signaling Technology, Beverly, MA, 1:500 dilution) monoclonal antibodies. The proteins were detected with enhanced chemiluminescence reagents.
For the IF studies, cells were fixed with 4 % paraformaldehyde in phosphate-buffered saline and permeabilized with 0.2 % Triton X-100 in phosphate-buffered saline. Fixed cells were incubated with 1:2000 fluorescein isothiocyanate–conjugated phalloidin (Sigma, St. Louis, MO) or antibodies as indicated. Cells were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (Calbiochem, San Diego, CA) and imaged with a confocal laser-scanning microscope (Olympus FV1000, Tokyo, Japan).

For detection of the actin ring, mature osteoclasts were transduced with SELENOW-overexpressing retrovirus, fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and stained with fluorescein isothiocyanate-conjugated phalloidin (Sigma-Aldrich). Fluorescence images were acquired with a BX51 microscope (Olympus, Tokyo, Japan). TRAP + MNCs with a full actin ring were counted to assess osteoclast survival. To measure caspase activity in mature osteoclasts, osteoclast precursors were differentiated into osteoclasts in the presence of M-CSF and RANKL for 3 days. Cells were washed twice with PBS and treated with 0.1% trypsin-EDTA for 5 min to remove the monocytes. After washing with PBS, osteoclasts were incubated with enzyme-free cell dissociation solution (Millipore) for 30 min. Purified osteoclasts were re-plated at 1 × 10⁶ cells on 10-cm culture dishes and then incubated for 24 or 36 h with M-CSF and RANKL. Caspase activity was assayed with a fluorometric kit (R&D Systems, Minneapolis, MN, USA). Briefly, the cells were washed with ice-cold PBS and lysed in the cell lysis buffer provided with the kit. The caspase-3 [DEVE-p-nitroaniline (pNA)] or caspase-9 (LEHD-pNA) substrate was added to the lysates in a 96-well plate followed by incubation for 1 h. The release of pNA was measured at 405 nm on a microplate reader.

To evaluate the effects of HX on the morphometric parameters of C2C12 cells, 1 × 10⁴ cells per well were seeded in GM on coverslips. The plates were cultivated under both experimental conditions up to day-14 post-MD. The cells were fixed with 4% PFA for 10 min at RT then were washed in PBS twice for 10 min. the cells were permeabilized in 0.1% tween (Roth, Germany) for 5 min. After washing with PBS, the cells were incubated with 0.5 mg/mL fluorescein isothiocyanate conjugated phalloidin (Sigma-Aldrich, Germany diluted 1:30 in PBS) in dark for 30 min. Cell nuclei were counterstained with DAPI for 5 min. The cells were mounted with DABCO Mowiol and then were photographed under fluorescent microscope. Morphometric analysis of the myotubes indicating MD was conducted on five randomly chosen microscopic fields per experimental group (n = 6). The data parameters including number, length and size of the myotubes were measured using ImageJ software.

After guinea pigs were anesthetized using an intraperitoneal injection of a mixture of medetomidine (0.3 mg/0.3 mL/kg), midazolam (4 mg/0.8 nL/kg), butorphanol (5 mg/mL/kg), and saline (2.9 mL/kg), the head was promptly decapitated and the temporal bone was removed. The cochlea was extracted from the temporal bone and fixed with 4% PFA for 24 h at room temperature. The organ of Corti was excised, permeated with 0.3% Triton X-100 for 10 min, and stained with fluorescein isothiocyanate-conjugated phalloidin (1 µg/mL PBS; Sigma) for 1 h. Samples were washed with PBS and observed using fluorescence microscopy. The outer hair cells between 45% and 70% of the distance from the apex to the basal rotation (second rotation of the cochlea) were observed. The reason for observing the outer hair cells in this region is that the outer cochlea forms the strongest obstacle to rotation because of the octave band noise centered at 4 kHz (Viberg & Canlon, 2004 (link)).