Fixing cells: Difference between revisions
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== ''' | == '''Fixation''' == | ||
This can work well for fixing of mammalian cells in preparation for immunolabeling and microscopy. Depending on the epitope, different fixatives may be required; membrane-bound epitopes may do better without permeabilization. On the other hand, some antibodies are only able to recognize a protein epitope in its native form and will only work on frozen tissues because formaldehyde cross-linking denatures the protein. | |||
'''Reagents''' | '''Reagents''' | ||
-Fixation Solution: 4% Formaldehyde in PBS | -Fixation Solution: 4% Formaldehyde in phosphate buffered saline (PBS) solution, pH 7.4 - 7.6 (verify) | ||
-Permeabilization Solution: 0.1% Triton X-100 in PBS | -Permeabilization Solution: 0.1% Triton X-100 in PBS | ||
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2) For permeabilization, remove Formaldehyde Solution, and incubate cells in Permeabilization Solution for 5 minutes at room temperature. | 2) For permeabilization, remove Formaldehyde Solution, and incubate cells in Permeabilization Solution for 5 minutes at room temperature. | ||
3) Rinse in PBS before proceeding. | |||
For further information about fixing cells, see [[Immunocytochemistry]]. | For further information about fixing cells, see [[Immunocytochemistry]]. | ||
==External Links== | |||
[http://www.chemicon.com/techsupp/AcetoneMethod.asp Acetone Fixation protocol] | |||
[[Category:Protocol]] [[Category:Microscopy]] | |||
Latest revision as of 11:42, 9 May 2007
Fixation
This can work well for fixing of mammalian cells in preparation for immunolabeling and microscopy. Depending on the epitope, different fixatives may be required; membrane-bound epitopes may do better without permeabilization. On the other hand, some antibodies are only able to recognize a protein epitope in its native form and will only work on frozen tissues because formaldehyde cross-linking denatures the protein.
Reagents
-Fixation Solution: 4% Formaldehyde in phosphate buffered saline (PBS) solution, pH 7.4 - 7.6 (verify)
-Permeabilization Solution: 0.1% Triton X-100 in PBS
Protocol
1) For fixation, incubate cells in Formaldehyde Solution for 10-15 minutes at room temperature.
2) For permeabilization, remove Formaldehyde Solution, and incubate cells in Permeabilization Solution for 5 minutes at room temperature.
3) Rinse in PBS before proceeding.
For further information about fixing cells, see Immunocytochemistry.
