MCF-10A cells were cultured to confluence and fixed with 4% (wt/vol) paraformaldehyde (PFA) in cytoskeleton stabilization buffer (10 mM piperazine-N,N’-bis(2-ethanesulfonic acid [PIPES] at pH 6.8, 100 mM KCl, 200 mM sucrose, 2 mM ethylene glycol-bis(b-aminoethyl ether)-N,N,N’,N’-tetraacetic acid [EGTA], 2 mM MgCl2) on ice for 20 min. Alternatively, cells were fixed for 10 min in –20°C MeOH at -20°C. PFA-fixed cells were permeabilized for 5 min in 0.25% Triton X-100 in phosphate-buffered saline (PBS). Cells were then blocked for 1 h on ice using 3% bovine serum albumin (BSA) in PBS, followed by incubation with primary antibodies diluted in 3% BSA in PBS overnight at 4°C. Cells were washed in PBS and then incubated with similarly diluted secondary antibodies for 90 min at room temperature before final washing in PBS. Coverslips were mounted onto slides using ProLong Gold containing DAPI (Cell Signaling Technologies; catalogue#8961). Immunostaining for phosphorylated proteins was performed using tris-buffered saline (TBS) instead of PBS.
Prolong gold containing dapi
Manufactured by Cell Signaling Technology
Prolong Gold containing DAPI is a mounting medium for fluorescence microscopy. It is designed to preserve fluorescent signals and counterstain nuclei with DAPI.
Automatically generated - may contain errors
4 protocols using prolong gold containing dapi
1
Immunofluorescence Staining of MCF-10A Cells
2
Immunofluorescence Staining of Kidney Cells
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Cells and kidney tubules were fixed using 4% PFA solution (Pierce™ 16% formaldehyde (w/v), methanol-free, ThermoFisher, Waltham, MA, USA) and subsequently permeabilized in 0.3% (v/v) Triton X-100 in HBSS. Kidney tubules were incubated with a blocking solution (2% (w/v) bovine serum albumin (BSA) fraction V and 0.1% (v/v) Tween-20 in HBSS) to prevent the non-specific binding of antibodies. Next, kidney tubules were incubated with the corresponding primary antibodies diluted in blocking solution overnight at 4 °C on a rocking platform. After washing off the primary antibodies, incubation with the secondary antibodies at RT for 2h was followed. Lastly, cells were incubated with Hoechst 33342 (dilution 1:10000) and kidney tubules were mounted using Prolong gold containing DAPI (Cell Signaling Technology, Leiden, The Netherlands) for nuclei staining. Images were acquired using the confocal microscope Leica TCS SP8 X (Leica Biosystems, Amsterdam, The Netherlands).
3
Evaluating Epithelial Integrity via Immunostaining
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To investigate epithelial-layer integrity, phenotypical and morphological characteristics, immunostainings were performed. Cells were fixed using 2% paraformaldehyde for 15 min and then permeabilized using 0.3% (v/v) Triton X-100 in HBSS for 5 min. To prevent non-specific binding of antibodies, blocking was performed using 2% (w/v) bovine serum albumin (BSA) fraction V and 0.1% (v/v) Tween-20 in HBSS for 1 h. Fixed cells were incubated with primary antibodies, diluted in block solution, used were tight junction protein, zonula occludens-1 (ZO-1, 1:400) (Thermo Fisher Scientific, Bleiswijk, The Netherlands), enterocyte marker, Villin (1:400) (SCBT, Texas, USA) for 4 h. This was followed by incubation with goat-anti-mouse Alexafluor 488 (1:1,000; Thermo Fisher Scientific) or goat-anti-rabbit CF640R (1:1,000) as secondary antibodies. Finally, HFM were mounted using Prolong gold containing DAPI (Cell signaling technology, Leiden, The Netherlands) for nuclei staining. Images were acquired using the Leica TCS SP8 X (Leica Biosystems, Amsterdam, The Netherlands).
4
Immunofluorescent Staining of Bioengineered Intestinal Tubules
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Immunofluorescent staining of the bioengineered intestinal tubules has been described before [14 (link)]. In short, cells were fixated (60% EtOH, 30% chloroform and 10% acetic acid (v/v)) for 5 min, permeabilized (0.3% (v/v) Triton X-100 in HBSS) for 10 min and blocked ((2% (w/v) bovine serium albumin (BSA) fraction V and 0.1% (v/v) Tween-20 in HBSS) for 30 min. Thereafter, bioengineered intestinal tubules were incubated with zonula occludens-1 (ZO-1) antibody (1:1000 diluted in blocking solution) (Thermo Fisher Scientific, Bleiswijk, The Netherland) for 2 h. After washing, a secondary antibody goat-anti-rabbit 594 (1:200) (Abcam, Cambridge, United Kingdom) was added. Finally, bioengineered intestinal tubules were mounted using Prolong gold-containing DAPI (Cell signaling technology, Leiden, The Netherlands) for nuclei staining. Images were acquired using the Leica TCS SP8 X (Leica Biosystems, Amsterdam, The Netherlands) confocal microscope. Immunofluorescent images are shown as maximum intensity projections of a z-stack.
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