Cells were cultured on glass coverslips in six-well plates, rinsed thrice with PBS, fixed with 3.7% paraformaldehyde for 15 minutes, and blocked with 5% normal goat serum for 1 hour. The cells were immunostained by using primary antibodies specific to various antigens. Goat anti-mouse IgG Alexa 488 or goat anti-rabbit IgG Alexa 546 was used as the secondary antibody. Images were taken under a Zeiss axiocam fluorescence microscope using Axiovision software (Zeiss, Germany).
Axiocam fluorescence microscope
Manufactured by Zeiss
Sourced in Germany
The AxioCam fluorescence microscope is a high-performance imaging tool designed for accurate and detailed fluorescence imaging. It features a high-resolution camera, advanced optics, and sophisticated software to capture clear and precise fluorescence images. The AxioCam is a reliable and versatile instrument suitable for a wide range of applications in scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Axiovision software, by Zeiss (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Eclipsete2000, by Zeiss (1 mentions)
Alexa fluor 594 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Anti p cadherin, by Cell Signaling Technology (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Merck Group (1 mentions)
Millicell ez slide eight well glass, by Merck Group (1 mentions)
4 protocols using axiocam fluorescence microscope
1
Immunofluorescence Staining of Cells
2
Immunofluorescence Analysis of E-cadherin and P-cadherin
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Cells were fixed for 20 min in 4% paraformaldehyde (Merck Life Science, Darmstadt, Germany) and blocked with 5% BSA (NZYTech) for 30 min. Cells were incubated with primary mouse antibody anti-E-cadherin (24E10) or anti-β-catenin (1:50; ON; 4 °C, Cell Signaling Technology, Danvers, MA, EUA) and primary rabbit monoclonal antibody anti-P-cadherin (#2130; 1:50; ON; 4 °C, Cell Signaling Technology), followed by secondary antibodies Alexa Fluor 488 Donkey Anti-Mouse (1:500; 60 min; Life Technologies) or Alexa Fluor 594 anti-rabbit (1:500, 60 min; Life Technologies). Cell nuclei were stained with DAPI (1 μg/mL in PBS; 5 min incubation; Sigma, Merck Life Science, Darmstadt, Germany). All coverslips were mounted using Vectashield mounting media (Vector Laboratories, Newark, CA, USA) and cells were analysed by fluorescence microscopy (Imager.Z1, AxioCam fluorescence microscope or Eclipse TE-2000, both from Zeiss, Oberkochen, Germany) using AxioVision software (Rockville, White Plains, NY, USA).
3
Immunofluorescent Staining of Cells
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Cells were plated onto cover slips and grown overnight at 37°C in 5% CO2 in growth medium. Prior to staining, the cover slips were washed with PBS, fixed in 4% PFA for 10 minutes and permeabilized with 0.1% Triton X in PBS for five minutes. The coverslips were then blocked in 5% goat serum (Sigma-Aldrich, USA) in PBS and then incubated with primary antibody at room temperature. Antigens were detected using fluorescently labeled secondary antibodies. To stain for F-actin, a fluorescent conjugate of phalloidin was used (ThermoFisher Scientific, USA). Cover slips were then mounted onto microscope slides using a drop of ProLong Gold antifade reagent with DAPI (4,6-diamidino-2-phenylindole) (Invitrogen, USA). Slides were visualized using a Zeiss AxioCam fluorescence microscope.
4
Immunofluorescence Staining of ER, PR, and HER2
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Immunofluorescence staining was performed as previously done (17 (link)). Briefly, 5 * 104 cells were seeded in each well of Millicell EZ SLIDE eight-well glass (Millipore), fixed with 4% PFA, and permeabilized with 0.1% Triton X-100. After blocking with 2.5% goat serum, the cells were incubated with rabbit anti-human estrogen receptor α antibody (D8H8, 1:1,600, CST), rabbit anti-human progesterone receptor A/B (D8Q2J, 1:800, CST), or rabbit anti-human HER2/ErbB2 (D8Q2J, 1:400, CST) overnight at 4°C. After rinsing with phosphate-buffered saline (PBS), anti-rabbit IgG (Alexa Fluor 555 conjugate, CST) was used to incubate the cells for 30 min. Alexa Fluor 555-conjugated rabbit IgG (Alexa Fluor 555 conjugate, CST) was used as the negative control. The cells were then rinsed with PBS three times and stained with DAPI (CST) for 1 min. A Zeiss Axiocam fluorescence microscope was used for image acquisition and analysis.
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