I want to image liposomes, but without any bound peptide they do not stick to the Poly-L-Lysine coating of my coverslips. Has anyone tried to fixate liposomes? I would try 2-4 % PFA...
Thanks in advance for your input!
It depends completely on what the liposomes are composed of. Generally lipids are difficult to fix with PFA. For PFA fixation it would be best to have (primary) amines around.
People regularly use agarose to embed and immobilize liposomes for imaging, like in this paper: https://www.nature.com/articles/srep25254
Personally, I use 1.8 % methylcellulose to embed my liposomes for EM, but I don't know if that's applicable for light microscopy as well.
Thank you for your answer, Christina!
I could try agarose, but I also have to stain with antibodies and a lipophilic dye (Bodipy) afterwards. Although in the paper you shared they also stain, I wouldn't be able to wash away residual antibodies giving background...
Dear David,
Cold methanol fixation has traditionally been used before visualization of cytoskeletal elements. This method unacceptable for study of lipid droplets because it extracted the majority of cellular phospholipids and promoted fusion of lipid droplets. Cold acetone fixation is similarly unacceptable because the total cellular lipids are extracted, causing collapse of the shell of lipid droplet-associated proteins. Fixation of cells with paraformaldehyde is the method of choice, because the cells retain their lipid content and lipid droplet structure is unaffected. As more lipid droplet-associated proteins are already discovered and studied, it is better to use appropriate methods to avoid studying artifacts.
As I understand, another way, the guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol (BGTC) (bis (guanidinium)-tren-cholesterol) combined to the colipid dioleoyl phosphatidylethanolamine (DOPE) (dioleoyl phosphatidylethanolamine) has also shown to efficiently deliver the β-gal intracellularly without compromising its activity.
Ashish
Dear David,
For clarity again I send the repeat answer.
Cold methanol fixation has traditionally been used before visualization of cytoskeletal elements. This method unacceptable for study of lipid droplets because it extracted the majority of cellular phospholipids and promoted fusion of lipid droplets. Cold acetone fixation is similarly unacceptable because the total cellular lipids are extracted, causing collapse of the shell of lipid droplet-associated proteins. Fixation of cells with paraformaldehyde is the method of choice, because the cells retain their lipid content and lipid droplet structure is unaffected. As more lipid droplet-associated proteins are already discovered and studied, it is better to use appropriate methods to avoid studying artifacts.
As I understand, another way, the guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol (BGTC) (bis (guanidinium)-tren-cholesterol) combined to the colipid dioleoyl phosphatidylethanolamine (DOPE) (dioleoyl phosphatidylethanolamine) has also shown to efficiently deliver the β-gal intracellularly without compromising its activity.
Ashish
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