CaCl2, 11733457 and 1 MgCl2, bubbled with 95% O2/5% CO2, pH 7.4. Hippocampal coronal brain sections were cut using a vibratome filled with ice-cold ACSF. The slices were transferred at room temperature to a holding chamber and immersed in oxygenated ACSF, pH 7.4, for a stabilization period of 1 h before use. Dye Transfer Cells plated on glass coverslips were bathed with recording medium. The permeability mediated by gap junctions was tested by evaluating the transfer of LY that was microinjected into one cell to neighboring cells, as described previously. Glial Cell Communication in Nieman-Pick Type C The incidence of dye coupling was scored as the percentage of injections that resulted in dye transfer from the injected cell to more than one neighboring cell. The coupling index was calculated as the mean number of cells to which the dye spread in positive cases. In all experiments, dye coupling was tested by injecting a minimum of 10 cells. Dye uptake and Time-lapse Fluorescence Imaging Cell cultures. For time-lapse fluorescence imaging, astrocytes plated on glass coverslips were washed twice and were then exposed to Locke’s solution with 5 mM Etd. Fluorescence intensity was recorded in selected cells with ROIs in their nuclei. Images were captured every 30 s using a Q Imaging model Retiga 13001 fast-cooled monochromatic digital camera in an Olympus BX 51W1I microscope. Metafluor software was used for off-line image analysis and fluorescence quantification. To test for changes in slope, regression lines were fitted to points before and after various treatments using the Excel program. The mean values of the slopes were compared using 3 Glial Cell Communication in Nieman-Pick Type C GraphPad Prism software and expressed as AU/min. La3+ and Cx43 antibody were applied acutely or preincubated for 30 min, 2883-98-9 chemical information respectively. Acute slices. For “snapshot”experiments, acute slices were incubated with 20 mM Etd for 5 min in a chamber with oxygenated ACSF, pH 7.4. They were then washed five times for 2 min each with ACSF, followed by fixation at room temperature with 2% paraformaldehyde for 30 min. Finally, the slices were mounted in Fluoromount G and examined using a confocal laser-scanning microscope. The dye uptake ratio was calculated using ImageJ as the subtraction between the 17804190 fluorescence from representative cells and the background fluorescence measured where no labeled cells were detected. At least five fields were selected in each slice. dividing the 340-nm fluorescence image by the 380-nm image on a pixel-by-pixel base. Immunofluorescence and Confocal Microscopy For all immunostaining experiments, astrocytes grown on coverslips were fixed at room temperature in 2% paraformaldehyde for 30 min, washed three times with PBS, incubated in 0.1 M PBS-glycine three times for 5 min each, and rinsed in 0.1% PBS-Triton X-100 containing 10% NGS for 30 min. We first incubated cells for 2 h at room temperature with anti-GFAP polyclonal antibody diluted in 0.1% PBS-Triton X100 with 2% NGS. After three rinses in 0.1% PBS-Triton X-100, the cells were incubated for 50 min at room temperature with goat anti-rabbit Alexa Fluor 555. For Cx43 detection, cells were incubated with the anti-Cx43 monoclonal antibody for 1 h at room temperature. After three washes, cells were incubated for 50 min at room temperature with goat anti-mouse Alexa Fluor 488. After several washes, coverslips were mounted in Fluoromount G and examined using an upright microscope equipped with epifluo

This model focuses on the interaction between Hsf1 and Hsp90

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