Hello!
I am looking for some suggestion about the staining on acute rodent brain slices with tomato lectin in order to visualize with 2 photon microscopy the living microglia.
I am not sure about the staining protocol, I used to stain the slices in an interface chamber with 5- 10 ul/ml for 30 min and after one wash out of 20 min. After I see perfectly the blood vessels but not the microglia as well. Anyone can suggest me? I will appreciate so much
Thank you very much
Hello Giampaolo,
I usually use lectin to visualize blood vessel and for me microglia is an artefact staining.
If you want to visualize microglia, I suggest you to incubate more time your sample with lectin (i.e. overnight at 4°C, I see very well microglia).
Note that lectin doesn't like to much triton concentration in its incubation buffer and too long formalin fixation.
This book chapter give a good explanation of the different lectin staining for microglia:
https://www.researchgate.net/search.Search.html?type=publication&query=Tomato%20Lectin%20Histochemistry%20for%20Microglial%20Visualization&tabViewId=56d59086eeae391d52031443&previous=researcher
Good luck,
VM
You need to add cations to your buffer (PBS/TBS + cations) in order for the lectin to label microglia.
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