Characterization of iPSCs by Immunocytochemistry

[fa icon="calendar"] Apr 5, 2018 12:20:38 PM / by Zara Puckrin

As part of our contract services at REPROCELL, we offer induced pluripotent stem cell (iPSC) characterization by immunocytochemistry (ICC). Planning ICC can be tricky, as it requires careful selection of compatible antibodies and fluorochromes. However, if successful, the results are fantastic! Don’t waste valuable research time performing ICC, and let our experts do the work for you!

What pluripotency markers do you visualize?

At REPROCELL, we visualize a range of intracellular and cell surface pluripotency markers including OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, SSEA-3 and SSEA-4. Nuclei are counterstained with DAPI.

What antibodies do you use for iPSC characterization?

To target these markers, we use our own Stemgent® primary antibodies:

Stemgent® secondary antibody, Goat Anti-rabbit IgG Antibody (DyLight™ 488), can be used to visualise NANOG and OCT4. 

Do you offer double staining?

Yes, we also provide a double staining service where intracellular and cell surface markers may be visualised together.

Below are some examples of iPSCs we have characterized by ICC using single and double staining.

 

Figure 1: Characterization of fibroblast and blood derived iPSCs using single staining. These iPSCs were characterized by expression of intracellular pluripotency markers NANOG (A) and OCT4 (B). First row (A): Fibroblast derived iPSCs; Second row (B): Blood derived iPSCs. First column: Phase contrast image of iPSCs; Second column: Visualization of cell nuclei (blue: DAPI); Third column: visualization of intracellular markers (green: DyLight 488); Fourth column: merge of the second and third columns. All images were taken at 10x magnification.

A  Figure1Aphasecontrast Figure1Ablue Figure1Agreen Figure1Amerge
B  Figure1Bphasecontrast Figure1Bblue Figure1Bgreen Figure1Bmerge

 

Figure 2: Characterization of fibroblast derived iPSCs using single staining. These iPSCs were characterized by expression of cell surface pluripotency markers TRA-1-60 (A) and SSEA-4 (B). First column: Phase contrast image of iPSCs; Second column: Visualization of cell nuclei (blue: DAPI); Third column: Visualization of cell surface markers (red: Alexa Fluor 594); Fourth column: Merge of the second and third panels. All images were taken at 10x magnification.

A  Figure2Aphasecontrast Figure2Ablue Figure2Ared Figure2Amerge
B  Figure2Bphasecontrast Figure2Bblue Figure2Bred Figure2Bmerge

 

Figure 3: Characterization of fibroblast derived iPSCs using double staining. These iPSCs were characterized by expression of cell surface marker TRA-1-60 and intracellular marker NANOG. Clockwise from top left: Cell nuclei (blue: DAPI); TRA-1-60 (red: Alexa Fluor 594); NANOG (green: DyLight 488); phase contrast; merge of TRA-1-60 and NANOG; merge of nuclei and NANOG. All images were taken at 10x magnification.

Figure3blue Figure3red Figure3green
Figure3redbluemerge Figure3redgreenmerge Figure3phasecontrast

 

Figure 4: Characterization of fibroblast derived iPSCs using double staining. These iPSCs were characterized by expression of cell surface marker SSEA-4 and intracellular marker OCT4. Clockwise from top left: Cell nuclei (blue: DAPI); SSEA-4 (red: Alexa Fluor 594); OCT4 (green: DyLight 488); Phase contrast; Merge of SSEA-4 and OCT4; Merge of nuclei and OCT4. All images were taken at 20x magnification.

Figure4blue Figure4red Figure4green
Figure4bluegreenmerge Figure4redgreenmerge Figure4phasecontrast

Topics: Drug Discovery, Stem Cells

Zara Puckrin

Written by Zara Puckrin

Zara Puckrin, Student Intern at REPROCELL
Zara is currently a B.Sc. student at Glasgow Caledonian University, UK, studying Cell and Molecular Biology.