After fixation with 4% paraformaldehyde, slides were placed in blocking solution [10% normal goat serum and 0.3% Triton X-100 in phosphate-buffered saline (PBS)] for 45 min at room temperature. Primary antibody staining was done at 4°C overnight in PBS with 5% normal goat serum and 0.1% Triton X-100 (The Dow Chemical Company), using LysoTracker fluorescent dye (L12492, ThermoFischer Scientific) and Cathepsin D antibody (2284, Cell signalling). The slides were then washed 3 times with PBS (0.1% Triton X-100 in PBS), incubated with secondary antibody (Invitrogen, 1:500) for 1 h at room temperature in the dark, and then washed 3 times before drying and adding Vectashield/4′,6-diamidino-2-phenylinodole stain (Vector Laboratories). Coverslips were mounted with Permount (Fisher Scientific). The cells were imaged and acquired using the PANNORAMIC 250 fluorescent microscope scanner (3DHISTECH). An average of 100 cells from 3 independent experiments per each condition were analysed by a blinded investigator.
Pannoramic 250 fluorescent microscope scanner
Manufactured by 3DHISTECH
The PANNORAMIC 250 is a fluorescent microscope scanner manufactured by 3DHISTECH. It is designed to digitize fluorescently labeled tissue samples for high-throughput imaging and analysis.
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Lab products found in correlation
2 protocols using pannoramic 250 fluorescent microscope scanner
1
Intracellular Organelle Visualization Assay
2
Lysosomal Protein Imaging in Cells
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After fixation with 4% paraformaldehyde, slides were placed in blocking solution (10% normal goat serum and 0.3% Triton X-100 in phosphate-buffered saline [PBS]) for 45 minutes at room temperature. Primary antibody staining was done at 4°C overnight in PBS with 5% normal goat serum and 0.1% Triton X-100 (The Dow Chemical Company), using LysoTracker fluorescent dye (L12492, ThermoFischer Scientific) and Cathepsin D antibody (2284, Cell signaling). The slides were then washed 3 times with PBS (0.1% Triton X-100 in PBS), incubated with secondary antibody (Invitrogen, 1:500) for 1 hour at room temperature in the dark, and then washed 3 times before drying and adding Vectashield/4′,6-diamidino-2phenylinodole stain (Vector Laboratories). Coverslips were mounted with Permount (Fisher Scientific). The cells were imaged and acquired using the PANNORAMIC 250 fluorescent microscope scanner (3DHISTECH). An average of 100 cells from 3 independent experiments per each condition were analysed by a blinded investigator.
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