Question

I am wondering whether we can replace 70% ethanol to fix and perm the cells with 4% PFA to observe the cell cycle. Do I understand correctly that 4% PFA can permeate the cells, thus the PI will intercalate DNA?

Answer 1

70% ethanol and 4% PFA are both used to fix and permeabilize cells; 70% ethanol is used for antibody permeability in ICC, while 4% PFA preserves cellular structure and enables PI to intercalate DNA. Both reagents serve to prepare cells for imaging but need careful consideration for compatibility with experimental protocols.

Step-by-step explanation:

When considering methods for cell cycle analysis, it's important to understand the role of different chemicals in the process. While 70% ethanol is commonly used in a technique called Immunocytochemistry (ICC) to make the cell membrane permeable and allow the entry of antibodies, it also acts to fix and permeabilize the cells. The treatment with alcohol makes holes in the cell membrane sufficiently large enough for antibodies to pass through, as mentioned in a similar context.

On the other hand, 4% paraformaldehyde (PFA) is a chemical fixative that also permeabilizes cells. It cross-links proteins, which helps to preserve the cellular structure and can facilitate the entry of dyes such as propidium iodide (PI) into the cells to intercalate DNA. Therefore, it is indeed possible to use 4% PFA to fix and permeabilize cells for cell cycle observation, allowing PI to intercalate with the DNA.

However, the compatibility and effectiveness of reagents can depend on the specific experimental conditions, so it is important to consult relevant protocols or literature to ensure that the substitution will yield reliable results.