Prior to treatment, cells were seeded on glass coverslips. Following treatment, cells were washed in PBS, fixed for 15 minutes in 4% formaldehyde, and washed again. Cells were then permeabilized in 100% methanol at −20°C for 10 minutes, washed, and blocked in PBST containing 1% normal goat serum for 1 hour. Cells were then incubated with primary antibody overnight at 4°C, washed thrice with PBS, and then incubated for 2 hours at room temperature with Alexa Fluor secondary antibodies (Invitrogen). Cells were washed once, and then treated with dapi at 1∶3000 dilution in PBS for 15 minutes. Coverslips were mounted using Vectashield mounting media (Invitrogen), and visualized using an LSM710 Carl Zeiss confocal microscope.
Vectashield mounting media
Manufactured by Thermo Fisher Scientific
Sourced in Italy
Vectashield mounting media is a glycerol-based solution designed for use in fluorescence microscopy. It is formulated to preserve fluorescent signals and protect samples from photobleaching.
Automatically generated - may contain errors
5 protocols using vectashield mounting media
1
Immunofluorescence Staining of Cultured Cells
2
Fluorescence Microscopy of Aptamer Interactions
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HEK293 and A549 cells were plated on cover slips in 6-well plates with DMEM containing 10% FCS. After 30 hours, the medium was removed and Dox, PS1-Zn-Dox and PS2-Zn-Dox containing DMEM medium were added to the cells. After 8 h, the cells were washed with chilled PBS solution twice, further fixed in 4% paraformaldehyde at RT for 10 min and mounted with Vectashield mounting media (Invitrogen). The PS2-Zn-aptamer was also used for microscopic studies in a separate set of experiments. Then cells were visualized by fluorescence microscopy (Optika B-100FL HBO, Italy) for labelled fluorescence.63 (link)
3
Fluorescence Microscopy of MCF7 Cells
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Cover slips were placed in six-well plates and MCF7 cells were grown on them using DMEM enriched with 10% FCS. The treatment of SN and SN-Dox compounds at a concentration of 100 nM in DMEM was done after a 30 hrs incubation period. Following an 8 hrs incubation time with the synthesized compounds, the cells underwent three consecutive 5-minute rinses with a chilled PBS solution. Next, the cells were fixed at room temperature for 10 minutes using 4% paraformaldehyde and subsequently washed twice with cold PBS. The cells were then mounted with Vectashield mounting media (Invitrogen) and subjected for the examination using fluorescence microscopy (Optika B-100FL HBO, Italy) to facilitate the visualization of the labeled fluorescence.65 (link)
4
Immunocytochemical Analysis of Breast Cancer Cells
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For immunocytochemistry 2 x 105 MDAMB-231 cells were seeded in 6 well plates on cover slips. After 24 hrs, cells were transfected with miR-200a, miR-200b, miR-15a, miR-429 and miR-203. Scramble miRNA vector was transfected into cells used as control. 48 hrs post-transfection, cells were fixed using 4% para-formaldehyde followed by three washes of 5 minutes each in PBST (Phosphate Buffered Saline with Tween® 20). Slides were further incubated overnight at 4°C separately with the following primary antibodies: anti-rabbit-BMI1 (Cell Signaling cat #. 6964), anti-rabbit-RING1A (Cell Signaling cat #. 13069 1:1000 dilution), anti-rabbit-RING1B (Cell Signaling cat #. 5694), anti-mouse-N-Cad (Cell Signaling cat #. 14215), anti-rabbit-Vimentin (Cell Signaling cat #.5741), anti-rabbit-ABCG2 (Cell Signaling cat #.4477),anti-rabbit-Ki67 (Cell Signaling cat #.9129). PBST washes were repeated for three times of 5 minutes interval each. Slides were thereafter incubated with the anti-mouse-FITC anti-rabbit-CY3 secondary antibody (Jackson Immuno Research) for two hours and finally washed in PBS, dried and mounted with Vectashield mounting media containing DAPI that counter stains the nucleus (Invitrogen, cat. #D21490). Slides were viewed under confocal microscope (Olympus Model no FV1000).
5
HEK293 Cell Fluorescence Microscopy
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For this experiment, we used HEK293 cells plated on sterile cover slips in 6 well plates with DMEM containing 10% FCS. After 24 h, the medium was removed and test compounds containing DMEM medium were added. After 6 h, the cells were washed three times with chilled PBS, fixed in 4% paraformaldehyde at RT for 10 min and mounted using Vectashield mounting media (Invitrogen). Cells were then analyzed by fluorescence microscopy (Optika B-100FL HBO, Italy) for morphological analysis and labelled fluorescence.
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