After 72 hours of non–T- or PAICS-siRNA transfection, the RT112 cells were seeded in chamber slides (Lab-Tek II CC2, Nunc, Rochester, NY) and incubated for a day to immunostain with rabbit monoclonal E-cadherin antibody (IF, 1:200; catalog #3195, Cell Signaling Technology, Danvers, MA). The slides were washed with phosphate-buffered saline (PBS) and were fixed using ice-cold methanol. Following three times PBS with 0.05% Tween 20 (PBS-T) wash, the slides were blocked for 2 hours using 5% normal horse serum in PBS-T. A rabbit anti-E-cadherin antibody was added to the slides at 1:200 dilution and incubated for 1 hour at room temperature. Following three times PBS-T wash, the slides were incubated with Alexa 555–conjugated goat, anti-rabbit antibody (Invitrogen) for 1 hour in the dark at room temperature. After three times PBS wash, the slides were mounted using ProLong Gold Antifade Mountant with DAPI (catalog #P36931, Invitrogen, ThermoFisher Scientific, Carlsbad, CA). Confocal images were taken with a 60× lens Nikon A1 High Speed Laser Confocal Spectral Imaging microscope (Nikon Instruments Inc., Melville, NY) from UAB high-resolution imaging facility.
Lab tek 2 cc2
Manufactured by Thermo Fisher Scientific
The Lab-Tek II CC2 is a laboratory equipment designed for cell culture applications. It provides a controlled environment for the growth and maintenance of cells, ensuring consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 555 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
E cadherin rabbit monoclonal antibody, by Cell Signaling Technology (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Oil red o, by Merck Group (1 mentions)
Element software, by Nikon (1 mentions)
Eclipse 80i epifluorescence microscope, by Nikon (1 mentions)
Vectashield mounting solution, by Vector Laboratories (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
3 protocols using lab tek 2 cc2
1
Immunostaining of E-cadherin in RT112 Cells
2
Lipid Staining of Fibroblasts
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Freshly isolated FBs were plated on a chamber slide (Lab-Tek II CC2, Nunc) and cultured for 72 h, then fixed in 4% paraformaldehyde for 20 min at room temperature. Cells were stained with 0.18% weight/volume Oil Red O (Sigma) in 60% isopropanol for 30 min, then counterstained with hematoxylin for 1 min.
3
Fluorescence Imaging of CAIX Expression
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CAIX-positive SK-RC-52 and CAIX-negative BxPC3 cells were seeded on to 8-well chamber glass slides (Lab-Tek®IICC2™, Nunc, Rochester NY) and incubated in RPMI 1640 media supplemented with 10% FBS and 1 × penicillin-streptomycin in a 37°C humidified incubator for 48 h. Cells were stained with 100 nM of FITC-labeled 8 for 1 h in the same growth media followed by washing twice with the same media. Cells were fixed with 10% formaldehyde (Sigma-Aldrich, Saint Louis, MO) and washed three times with PBS. Cells were treated with 20 nM DAPI (4′,6-diamidino-2-phenylindole) in PBS. The chambers were removed and the Vectashield mounting solution (Vector Laboratories, Inc., Burlingame, CA) was added to the sample. Fluorescence microscopic images were taken using the Nikon Eclipse 80i epifluorescence microscope (Nikon Instruments Inc., Melville, NY) and the images were processed by the Element software (Nikon Instruments Inc.).
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