I am preparing a live/dead assay to know the viability of cells encapsulated in alginate hydrogels, using Hoechst/Propidium iodide double stain.
Are there any differences between using "formalin" or "glutaraldehyde" for cell fixation encapsulated in the hydrogel for immunofluorescence?
Thanks, Amin,
Although it was a really informative article, I emphasize on fixation of cells encapsulated in "hydrogels". When cells are spreading on plastic surfaces, I normally use GA. I wanted to know what is the difference between these two conditions since for hydrogels, using formaldehyde is recommended.
It is preferred to use formaldehyde with hydrogels for immunofluorescence since in my experience glutaraldehyde shows a lot of autofluorescence which interferes with the imaging of cells. Typically for immunofluorerscence of cells on gels (hydrogels/cryogels) I use 4% paraformaldehyde for fixing and it gives really good results.
Dear Apeksha,
thank you for your attention. As you mentioned, when I used the glutaradehyde, I observed autofluorescence from the hydrogel, too. Actually I thought it might be because of dye concentration, but before that, I think I should try formaldehyde or paraformaldehyde ASAP. Thanks for your helpful comment.
Apeksha Damania Did you use fluoresent antibody to stain cells in hydrogel after fixation? I'm trying to stain cells in hydrogel by immunofluoresent assay.
Yoo-Jung Lee: Yes I have...on thin sections (approx. 100 micron) of cryogels...for immunofluorescence of albumin...what is your question, though?
Apeksha Damania I'm gonna try to stain neuronal markers in cells encapsulated hydrogel. But I don't know cells in hydrogel can be stained directly without cryosection. Our hydrogel is too fragile, so I want to stain cells in hydrogel directly.
Yoo-Jung Lee- how thick is your hydrogel? Is it opaque or transparent? If it is not a very thick sample (anywhere between 100-200 micron) it will be possible to stain the cells. Also, if the hydrogel is transparent or translucent, visualization of the stain under the microscope would be relatively better. In case your hydrogel is very thick (say in mm range), you may want to first fix the cells, and then try to take cryosections. If the hydrogel is fragile, cryosections of 60-80 microns may not be possible, hence you may want to increase the section thickness to around 100-200 microns. Accordingly, you will have to increase your cell seeding density.
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