I am planning to do colacalisation study between nanoparticle and endosome and lysosome. I am looking into various options to label endosome and lysosome. options available are as follows: for endosome labelling- using transferrin, and for lysosome- using LAMP-1 or lysotracker. I would appreciate if anyone who have already done this work can advice what will be suitable marker for me to use. and pro and cons of using them. thank you.
If you plan to do live-cell-imaging, Tf and lysotracker would work fine. Just be aware that Tf will stain the cell surface first, then endosomes (after about 5-10 min), and may also traffick all the way back to the trans-Golgi network (20-30 min).
If you fix the cells, you can do immunestaining for more specific endosomal markers, like EEA1, Rab 6 or Rab 11 for early, late and recycling endosomes, respectively. LAMP1 is a classic lysosomal marker and will work fine.
If you plan to do live-cell-imaging, Tf and lysotracker would work fine. Just be aware that Tf will stain the cell surface first, then endosomes (after about 5-10 min), and may also traffick all the way back to the trans-Golgi network (20-30 min).
If you fix the cells, you can do immunestaining for more specific endosomal markers, like EEA1, Rab 6 or Rab 11 for early, late and recycling endosomes, respectively. LAMP1 is a classic lysosomal marker and will work fine.
Hi Margarethe. I plan to fix my cells. I am planning to co-incubate transferrin and nanoparticle and fix the cells once the incubation time is done. for lysotracker, I plan to stain the cells after incubation with nanoparticle and proceed for fixing the cells. what do you think about this? thank you
It depends on what type of endosomes you want to label. There are early endosomes, late endosomes and recycling endosomes. There are different markers available for different endosomes like Rab5 is early endosome marker, Rab7 is late endosome marker and Rab11 is recycling endosome marker. Either you can use commercial antibodies against these porteins and if there are no good antibodies available for these proteins then either you can transfect your cells with these protein constructs or you can use good antibodies against some other proteins that are transported by these endosomes.
I would suggest, the excellent anti cathepsin D antibody (goat polyclonal) sold by Santa Cruz. to label lysosomes. It works very well on formol fixed material, at low concentrations and has therefore a very good quality/price ratio. I have published recently micrographs of confocal studies with this and other markers of the lysosome autophagy system.
It depends to what typre of endocytosis pathways you are looking for. Because there are a number of endosme markers start from endosytosis ligands (transferrin as a clathrin markers or dextran as a macropinocytosis markers )to a marker of endocytosis compartments ( rab5 or EEA as an early endosomal compartmentsor LAMP1 a lysosomal marker ). But if you want to do colocalization is recomnded to use a Z stake image and confocal microscopre is a best choice for you.
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