I used formalin-fixed paraffin-embedded liver tissue to do the immuofluorescent staining, but the result doesn't look good, can anyone tell me what is possibly wrong with it?
I used 20% normal goat serum and 0.15% glycine to block, the buffer I used is TBST.
Hello
Picture is not clear for me .
But most protocols using formalin or formaldehyde fixation its require a second permeabilization step, using either detergents (ranging from mild to moderate, saponin/digitonin, 0.1-0.5% Triton-X100, NP40) or organic solvents such as methanol or acetone.
Hope help you
http://www.biotechniques.com/multimedia/archive/00074/CRI-FP-Microscopy_74545a.pdf
Hello Pete Tu,
It would be better if you could provide staining information in detail. Like, your primary and secondary antibody dilution rate, dilution buffer, time of incubation, incubation temperature, host of antibody, and also washing steps. For blocking you should consider host of antibody you are using.
Hello
Permeabilization should only be required for intracellular epitopes when the antibody required access to the inside of the cell to detect the protein. However, it will also be required for detection of transmembrane membrane proteins if the epitope is in the cytoplasmic region.
Was the mouse perfused with cold ringer or PBS and the fixative?
If the tissue is not correctly perfused you could have some autofluorescence from the blood vessels.
Hi, I usually add 0.3% of Triton X 100 in my blocking buffer to permeabilize my cells. BTW the 'T - tween-20' in your TBST is a permeabilizing agent too.
I think that a lot of the background you have in your staining is due to the autofluorescence of the vessels. You can actually see them (the small green lines you see among your cells).Try to perrfuse the mice with cold ringer solution and PFA 4% ( it's a common protocol you can easily find in literature). I hope it can help you.
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