Fixing cells

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Current revision (14:42, 9 May 2007) (view source)
 
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== '''Formaldehyde Fixation''' ==
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== '''Fixation''' ==
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*This works well for fixation of mammalian cells in preparation for immunolabeling and microscopy
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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'''
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-Fixation Solution: 4% Formaldehyde in PBS
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-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.
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3) Rinse in PBS before proceeding.
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For further information about fixing cells, see [[Immunocytochemistry]].
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==External Links==
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[http://www.chemicon.com/techsupp/AcetoneMethod.asp Acetone Fixation protocol]
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[[Category:Protocol]] [[Category:Microscopy]]

Current revision

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.

External Links

Acetone Fixation protocol

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