I've been imaging fibroblasts 'eating'/engulfing particles and they seem to be stored/held within small spherical structures surrounding the nucleus. How can I best identify what these compartments are? Antibodies against endosomes, lysosomes, golgi etc - is this the way to go? Any advice gratefully received.. Thanks!
Immunofluorescence is a good idea, indeed. You might want to check the Molecular Probes website for what they have to offer (for fixed/live cells): http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Cell-Staining-Tool.html
Are your particles somehow tagged, e.g. His-tag, Strep-tag or similar? If yes, you can also attempt to fractionate a cell lysate and look in which cellular compartment they reside, using antibodies against different compartments to identify each compartment.
I agree with Maria. ICC with specific ABs is a good choice. There are several markers for distinct cellular compartments available. There are several nice markers from CST vailable. TGN38 for Golgi, Lamp2 for lysosomes, e.g.
However, you can also use fluorescent dyes that directly label your compartments of interest such as Mito-tracker, Lyso-tracker and so an (from Molecular probes).
Hope that helps...
Thanks all! Very helpful..
Has anyone had experience using an of the Rab antibodies, for example Rab5 and Rab7? And if so - any recommendations of where to source them?
You could use a cell-tracker that stains membranes - then "engulf" your particles and check the spherical structures for membrane remnants. Or use a membrane-stain or a tracker with a different colour if the particles already contain a membrane.
Http://www.ncbi.nlm.nih.gov/pubmed/9062793 an elegant but sophisticated way often used in neuroanatomy
From your description, it sounds like lipid droplets. They are normally found on ER membrane surrounding nucleus. You could use any LipidTox dyes or Nile Red to identify them.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology