EMS and healthy cells were first seeded onto coverslips (Zeiss, Oberkochen, Germany) and pre-treated with C. glomerata extract at 0.5% and 1% for 24 h. The remaining medium was after that washed off; cells were rinsed using HBSS and fixed in 4% paraformaldehyde for 40 min at room temperature. Subsequently, the cell membranes were permeabilized with 0.1% Triton X-100 for 15 min at room temperature, washed three times with HBSS, and incubated 45 min in blocking buffer containing 10% Goat Serum and 0.2% Tween-20 in HBSS. Primary antibodies against Ki-67 (Abcam, Cambridge, UK) (1:500 in HBSS containing 1% Goat Serum and 0.2% Tween-20) were then added to cells and incubated overnight at 4 °C. After washing of antibodies excess, cells were labeled with goat anti-mouse secondary antibodies conjugated with atto-488 (1:1000, Abcam, Cambridge, UK) for 1 h in the dark, at room temperature in a humidified chamber. The immunostained cells were finally mounted in ProLong Gold Antifade containing DAPI (Life Technologies, Warsaw, Poland) and were visualized and imaged using confocal microscope (Zeiss Cell Observer SD).
Goat anti mouse secondary antibodies conjugated with atto 488
Manufactured by Abcam
Sourced in United Kingdom
Goat anti-mouse secondary antibodies conjugated with atto-488 are designed for use in immunoassays and immunofluorescence microscopy. These antibodies specifically bind to the Fc region of mouse immunoglobulins, allowing the detection of mouse primary antibodies. The atto-488 fluorescent label enables visualization under a fluorescence microscope.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade containing dapi, by Thermo Fisher Scientific (2 mentions)
Cell observer sd, by Zeiss (2 mentions)
Primary antibodies against lamp2, by Abcam (2 mentions)
Observer z1 confocal spinning disc v 2 with live imaging chamber, by Zeiss (2 mentions)
Primary antibodies against ki 67, by Abcam (1 mentions)
4 protocols using goat anti mouse secondary antibodies conjugated with atto 488
1
Immunofluorescence of Ki-67 in Cells Treated with Calliandra glomerata Extract
2
Immunocytochemistry of LAMP2 in Cells
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EMS and healthy cells were first seeded onto coverslips (Zeiss, Oberkochen, Germany) and pre-treated with calystegines for 24 h. The remaining medium was after that removed; cells were washed with HBSS and fixed in 4% paraformaldehyde for 40 min at room temperature. Subsequently, the cells were permeabilized with 0.1% Triton X-100 for 15 min at room temperature, washed three times with HBSS, and incubated 45 min in blocking buffer containing 10% goat serum and 0.2% Tween-20 in HBSS. Primary antibodies against LAMP2 (Abcam, Cambridge, UK) diluted with 1:500 in HBSS containing 1% goat serum and 0.2% Tween-20 were then applied to cells overnight at 4 °C. After washing of antibodies excess, cells were treated with goat anti-mouse secondary antibodies conjugated with atto-488 (1:1000, Abcam, Cambridge, UK) for 1 h in the dark, at room temperature in a humidified chamber. The immunostained cells were finally mounted in ProLong Gold Antifade containing DAPI (Life Technologies, Warsaw, Poland) and were visualized and photographed using a confocal microscope (Zeiss Cell Observer SD).
3
Mitochondrial Network Visualization Protocol
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Prior MT visualization, cells were incubated with MitoRed dye (1:1000) in 37°C for 30 minutes, fixed with PFA and rinsed with HBSS. Nuclei were counterstained with diamidino‐2‐phenylindole (DAPI; 1:1000 in HBSS) for 5 minutes. For immunofluorescence, cells were fixed in 4% PFA and permeabilized with 0.5% Triton X‐100. Non‐specific binding sites were blocked with blocking buffer (10% Goat Serum, 0.2% Tween‐20 in HBSS) for 45 minutes. Cells were then incubated overnight at 4°C with primary antibodies against LAMP2 (Abcam) diluted 1:500 in HBSS containing 1% Goat Serum and 0.2% Tween‐20. After washing, samples were incubated for 1 hour with goat anti‐mouse secondary antibodies conjugated with atto‐488 (dilution 1:1000; Abcam). Nuclei were counterstained with DAPI. Specimens were observed and photographed using confocal microscope (Observer Z1 Confocal Spinning Disc V.2 Zeiss with live imaging chamber) and analysed using ImageJ software. Based on representative photographs, mitochondrial net was visualized in microP software.30
4
Visualizing Mitochondria and LAMP2 Localization
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In order to visualize the mitochondria, cells were incubated with MitoRed dye (1 : 1000) in 37°C for 30 minutes. Following fixation with PFA, cells were rinsed three times with HBSS and cells' nuclei were counterstained with diamidino-2-phenylindole (DAPI; 1 : 1000 in HBSS) for 5 minutes.
Prior to the analysis of LAMP2 localization, cells were fixed in 4% PFA for 30 min and washed three times with HBSS. Then, cells' membranes were permeabilized with 0.5% Triton X-100 for 20 min at room temperature. After washing with HBSS three times, unspecific binding sites were blocked with blocking buffer (10% Goat Serum, 0.2% Tween-20 in HBSS) for 45 min. Cells were then incubated overnight at 4°C with primary antibodies against LAMP2 (Abcam) diluted 1 : 500 in HBSS containing 1% Goat Serum and 0.2% Tween-20. Cells were then washed again and incubated for 1 hour with goat anti-mouse secondary antibodies conjugated with atto-488 (dilution 1 : 1000, Abcam), avoiding direct light. Subsequently, nuclei were counterstained by incubation with DAPI for 5 min. Cells were observed and photographed using confocal microscope (Observer Z1 Confocal Spinning Disc V.2 Zeiss with live imaging chamber) and analysed using ImageJ software.
Prior to the analysis of LAMP2 localization, cells were fixed in 4% PFA for 30 min and washed three times with HBSS. Then, cells' membranes were permeabilized with 0.5% Triton X-100 for 20 min at room temperature. After washing with HBSS three times, unspecific binding sites were blocked with blocking buffer (10% Goat Serum, 0.2% Tween-20 in HBSS) for 45 min. Cells were then incubated overnight at 4°C with primary antibodies against LAMP2 (Abcam) diluted 1 : 500 in HBSS containing 1% Goat Serum and 0.2% Tween-20. Cells were then washed again and incubated for 1 hour with goat anti-mouse secondary antibodies conjugated with atto-488 (dilution 1 : 1000, Abcam), avoiding direct light. Subsequently, nuclei were counterstained by incubation with DAPI for 5 min. Cells were observed and photographed using confocal microscope (Observer Z1 Confocal Spinning Disc V.2 Zeiss with live imaging chamber) and analysed using ImageJ software.
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