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Cell fixatives
Topics:
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The common fixative used in cytogenetics (usually a combination between methanol and acetic acid) helps keep the cells in a "swollen" state, achieved after hypotonic treatment. It also helps break the cell membrane of the red blood cells and elutes some of the membrane lipids and proteins. The fixative solution makes the cell membrane more fragile and suitable for spreading flat on the slide when subjected to the drying techniques.
Regular
3:1 fixative
The conventional 3:1 methanol:acetic acid fixative worked
well with all types of cells tested (lymphocytes, fibroblasts, tumor
cells). Quality of slide preparations as well as banding and FISH
depended more on the drying technique than on the proportion of methanol
to acetic acid. However, as detailed below, G-banding contrast was
influenced by the type of fixative (fig. 5,
6).
High
acetic acid
2:1 and 1:1 methanol:acidic acid concentrations were also
tested. Higher acid concentration generally improved spreading, probably
by increasing even more the fragility of the cell membrane and by
decreasing the speed of fixative evaporation. However, higher acetic
acid concentrations are NOT necessary if the drying technique is properly
mastered.
High
methanol (6:1) fixative
A 6:1 methanol acetic acid can be used to spread pellets that
tend to break too easily when conventional fixative and drying techniques
are used. By increasing methanol proportion, drying/evaporation of
the fixative is much faster, not allowing the cell membrane and the
content of the cells to spread too far.
Ethanol
fixative
3:1 ehanol:acetic acid was compared with the regular fixative.
Ethanol fixative worked well, metaphases were clean and the chromosomes
were prevented from overspreading, due in part to the slower evaporation
of ethanol compared to methanol. Apparently, a 3:1 ethanol:acetic
acid works similarly to a methanol:acetic acid fixative between 3:1
and 6:1. GTG banding and FISH quality using ethanol:acetic acid fixed
pellet was similar to regular fixative. Ethanol is much less toxic
than methanol and can be successfully used, particularly on pellets
that tend to overspread.