The cells were incubated on glass coverslips. After demonstrating appropriate attachment, the cells were fixed using 4% PFA in PBS. They were then washed with PBS and treated with phalloidin-594 and DAPI at room temperature for 1 h. After thorough washing, the cells on the coverslips were overlaid with a mounting medium (Cosmo Bio, Tokyo, Japan) and then analyzed using an FV1000 (Olympus, Tokyo, Japan).
Mounting medium
Manufactured by Cosmo Bio
Sourced in Japan
Mounting medium is a liquid solution used to prepare samples for microscopy. It functions to adhere and preserve specimens on a microscope slide, allowing for clear observation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using mounting medium
1
Immunofluorescence Staining of Cultured Cells
2
Immunofluorescence Staining of Cells
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Cells were incubated on glass coverslips. After demonstrating appropriate attachment, the cells were fixed using 4% paraformaldehyde in phosphate-buffered saline (PBS). They were then washed with PBS and treated with blocking buffer (5% goat serum and 0.3% Triton-X in PBS) at room temperature for 1 h and incubated overnight with anti-MITF antibody in antibody dilution buffer (1% bovine serum albumin (BSA) and 0.3% Triton-X in PBS) at 4 °C. The coverslips were washed three times with PBS and incubated with Alexa Fluor 488-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA, USA), phalloidin-594, and 4′,6-diamidino-2-phenylindole (DAPI) for 1 h. After thorough washing, the cells on coverslips were overlaid with mounting medium (Cosmo Bio), and then analyzed with an FV1000 (Olympus, Tokyo, Japan).
3
Immunofluorescence Staining of HS1 Protein
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Cells were incubated on glass coverslips. After demonstrating appropriate attachment, the cells were fixed using 4% PFA in PBS. They were then washed with PBS and treated with blocking buffer (5% goat serum and 0.3% Triton-X in PBS) at room temperature for 1 hour and incubated overnight with anti-HS1 antibody in antibody dilution buffer (1% BSA and 0.3% Triton-X in PBS) at 4° C. The coverslips were washed three times with PBS and incubated with Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA), phalloidin-594, and DAPI for 1 hour. After a thorough washing, the cells on coverslips were overlaid with mounting medium (Cosmo Bio), then analyzed with an FV1000 (Olympus).
4
Quantifying Invadopodia in siRNA-Transfected Cells
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NOE cells were transfected with siRNAs. After 48 hours, transfected NOE cells were detached and counted with the ViCell analyzer (Beckman Coulter, Tokyo, Japan). The cells were re-suspended and cultured overnight on gelatin-FITC (Elastin Products Company, Owensville, MO, USA) in the presence of 5-nM metalloproteinase (MMP) inhibitor (GM6001, Cayman Chemical, Ann Arbor, MI, USA). Supernatant was removed, the cells were washed with phosphate buffered saline, and culture medium without MMP inhibitor was added. After 6-hour culture, the cells were fixed and stained with phalloidin-594 (for F-actin; Thermo Fisher Scientific) and DAPI (for nuclei). The cells were then thoroughly washed, placed on glass slides, overlaid with mounting medium (Cosmo Bio), and analyzed using an FV1000 confocal laser scanning microscope (Olympus). In each image, the total number of cells and the number of invadopodia per cell were counted. The invadopodia were identified as gelatin degradation spots (black spots) that co-localized with F-actin (red spots). Invadopodia-positive cells were defined as having over 10 invadopodia. The percentage of invadopodia-positive cells was calculated as follows: [(total number of cells – number of invadopodia-positive cells) / total number of cells] × 100 (%).
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