Hello,
I need to fix my 3D cell culture (incubated with sperm cells), and after a pre-experiment I saw that the best fixative is the Karnovsky. But the auto-fluorescence rate is too high compared to Paraformaldehyde fixation. And I want to perform immunofluorescence assay with this cells.
I was wondering if there is any trick to reduce this auto-fluorescence when cells are fixed with Karnovisky.
Thank you!
Make up a 1 mg/ml solution of sodium borohydride in a physiological
buffer such as PBS. The solution will be fizzy like carbonated water. Preparing this solution on ice. Apply this solution immediately (while fizzing) to cells. Fixed cell monolayers incubate in the sodium borohydride solution for 4 minutes. Rinse many times with physiological saline to remove traces of sodium borohydride. Continue with blocking steps at this point. Discard any leftover sodium borohydride solution as it
loses its reactivity with time.
The use of it is particularly suited to reduce the reversible Schiff's bases that are formed by the aldehyde-NH2 reaction and lead to autofluorescence, especially when using glutaraldehyde and paraformaldehyde.
Karnovsky fixative can have variable % of glutaraldehyde. The higher the more auto-fluorescence is my experience. Try 0.5/1/1.5 and 2 %.
Hello
i agree with researchers
Karnovsky’s – this is a mixture of para-formaldehyde and glutaraldehyde, with post fixation in osmium tetroxide .
a- 5% glutaraldehyde, 4% formaldehyde in cacodylate buffer (cacodylic acid).
b- 2.5% GA, 2% paraformaldehyde
c- Cacodylate (0.2M) 25 ml
d- pForm (10%) 10 ml
e- GA (25%) 5 ml
f- distilled H2O to 50 ml
The mixture results in 0.1M buffered solution.
Depending on which cellular compartment is to be stained, alternative methods of fixation for immunofluorescence can be advantageous (e.g. ice-cold methanol for microtubule staining).Generally, the fixative to be used for 3D cultures should be determined by the nature of the antibody used and by its target rather than by the fact that the cultures are in 3D. A notable exception is acetone, which produces moderate swelling and should therefore be avoided.
Good Luck
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