MSCs were fixed in 4% PFA at room temperature for 15 min and permeabilized with 0.5% Triton X-100 for 5 min. The cells were blocked with 2.5% goat serum for 1 h and then stained with 1:200 anti-Ki-67 mouse monoclonal antibody (M7240; Dako; Agilent Technologies, Inc.) overnight at 4°C. Antibody binding was visualized by incubation with a FITC-conjugated goat anti-mouse secondary antibody (1:200, sc-2010; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Mouse IgG2a (1:200, sc-2856; Santa Cruz Biotechnology, Inc.) was used as the isotype control for Ki-67 antibody and FITC-conjugated goat anti-mouse antibody (1:200, sc-2010; Santa Cruz Biotechnology, Inc.) as a negative control to assess non-specific binding of the secondary antibody. MSCs were then stained with DAPI at room temperature for 3 min and imaged using an Olympus BX51 fluorescence microscope.
Fitc conjugated goat anti mouse antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
FITC-conjugated goat anti-mouse antibody is a secondary antibody that binds to mouse primary antibodies. The antibody is conjugated with the fluorescent dye FITC, which allows for the detection and visualization of mouse-specific proteins or targets in various applications such as immunofluorescence, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Fitc conjugated goat anti mouse secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse igg2a, by Santa Cruz Biotechnology (1 mentions)
Bx51 fluorescence microscope, by Olympus (1 mentions)
Ab955, by Abcam (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Ab6564, by Abcam (1 mentions)
Prolong gold mount gel, by Thermo Fisher Scientific (1 mentions)
Blockaid blocking solution, by Thermo Fisher Scientific (1 mentions)
Fv300, by Olympus (1 mentions)
4 protocols using fitc conjugated goat anti mouse antibody
1
Immunofluorescent Staining of MSCs
2
Quantitative Immunofluorescence Analysis of CD68
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prepare the sections for immunofluorescence analysis, non-specific binding to the sections was blocked by incubation at room temperature for 2 h with PBS containing 0.2% Triton X-100 and normal horse serum. The sections were incubated at 48°C overnight with a mouse monoclonal antibody against CD68 (1:200, ab955; Abcam, Cambridge, MA, USA). After incubation, the sections were washed three times for 5 min with 0.2% Triton X-100 in PBS, incubated at room temperature with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody (1:200, sc-2010; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min. The sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Quantitative data were derived from the immunofluorescent images by pixel analyses using ImagePro image analysis program (Image ProMedia Cybernetics, Rockville, MD).
3
Immunofluorescence Analysis of Epithelial Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were washed in PBS and fixed with 95% acetone for 15 min. Following fixation, the cells were washed in PBS, permeabilized with 0.25% Triton X-100 and 5% dimethylsufoxide (DMSO)-PBS for 10 min at room temperature and washed in PBS. Subsequently, 0.75/1.5% PBS-H2O2 was added to the cells for 15 min at 37°C, the cells were washed in PBS, and blocked in 10% sheep serum albumin (Abcam, Cambridge, UK). The cells were incubated overnight with a 1:200 dilution of the primary monoclonal antibodies (mAbs; mouse IgG, K19, involucrin and K1/10 Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) and subsequently incubated with the secondary antibody (FITC-conjugated goat anti-mouse antibody; Santa Cruz Biotechnology, Inc.) at 37°C for 30 min. Images were acquired using an Olympus BX51 microscope (Olympus, Tokyo, Japan) and the AnalySIS software, version 1.8 (Soft Imaging Systems, Olympus) and processed with Adobe Photoshop CS5 (Adobe, Mountain View, CA, USA).
4
Immunofluorescence Analysis of Tight Junction Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T84 cells were grown on Transwell filters (pore size 0.4 μm) and treated with ASP in HBSS supplemented with 15 mM HEPES, pH 7.4. After incubation for 6 hr at 37°C in a 5% CO2 atmosphere, the monolayer was washed three times in ice-cold phosphate-buffered saline (PBS) and fixed/permeabilized in ice-cold 100% ethanol at −20°C for 20 min. After being blocked with BlockAid Blocking Solution (Thermo Fisher Scientific, Waltham, MA), the samples were incubated with the primary antibodies against nectin-2 (ab135245, Abcam)1:250, afadin (A0349, Sigma-Aldrich)1:500, and E-cadherin (610181, BD Biosciences)1:500. The secondary antibody was Cy5-conjugated goat anti-rabbit antibody (ab6564, Abcam)1:1000 or FITC-conjugated goat anti-mouse antibody (sc-2010, Santa Cruz Biotechnology)1:200.
The samples were then washed three times with PBS and mounted in ProLong Gold mount gel (Thermo Fisher Scientific) with propidium iodide (PI; Nacalai Tesque). Fluorescence signals were visualized with a confocal laser scanning microscope (FV-300, Olympus, Tokyo). All experiments were performed in triplicate, and the reproducibility of the results was confirmed.
The samples were then washed three times with PBS and mounted in ProLong Gold mount gel (Thermo Fisher Scientific) with propidium iodide (PI; Nacalai Tesque). Fluorescence signals were visualized with a confocal laser scanning microscope (FV-300, Olympus, Tokyo). All experiments were performed in triplicate, and the reproducibility of the results was confirmed.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!