Hi all,
I want to stain NF-kB in HUVECs. Is it better to stain in paraformaldehyde or formaldehyde? Or are there no differences?
Thanks!
Paraformaldehyde is merely the polymerized (solid) form of formaldehyde. If you dissolve paraformaldehyde in fluid, you will have a solution of formaldehyde.
The main question you need to ask is whether you should use formaldehyde prepared in your lab from paraformaldehyde, or whether you should use a commercial solution of formaldehyde, which is usually called formalin, and which contains stabilizers like methanol. This is what I would recommend; a commercial formalin solution will be a lot more stable than something you prepare in the lab. The fact that it contains a small percentage of methanol is not a problem for the large majority of procedures, and should not affect the staining that you are trying to do; methanol is in itself a mild fixative at those concentrations. It can be a greater problem to use home-made formaldehyde that doesn't actually have the concentration that you think it has, because it hasn't been properly prepared or has precipitated in part.
The presence of methanol in the formaldehyde solution can have an impact.
Look at this old question with this link.
https://www.researchgate.net/post/Is_there_a_big_difference_between_using_formaldehyde_or_paraformaldehyde_for_cell_fixation_for_immunofluorescence
Formalin Fixation
Fix cells in 10% neutral buffered formalin for 5-10 minutes.
Rinse briefly with PBS.
Permeabilize with 0.5% Triton X-100 for 10 minutes.
Paraformaldehyde-Triton Fixation
Fix in 3-4% paraformaldehyde for 10-20 minutes.
Rinse briefly with PBS.
Permeabilize with 0.5% Triton X-100 for 10 minutes.
Paraformaldehyde-Methanol Fixation
Fix in 3-4% paraformaldehyde for 10-20 minutes.
Rinse briefly with PBS.
Permeabilize with cooled methanol for 5-10 minutes at –20 °C.
The effect of Paraformaldehyde fixation is much better than formalin fixation.
In addition to the fixation, the concentration of antibody needs to be
Tested carefully.
Fixation of Cells with 4% Paraformaldehyde
The following protocol provides information for the preparation of 3x PBS and 4% paraformaldehyde, which is used as a cell fixative. Freshly made the solution results in better signal. You can save the solution in plastic tube kept in freezer.
You can use it within one month.
Preparation of 3x PBS
1. Prepare two separate solutions composed of:
390 mM NaCl, 30 mM Na2HPO4
390 mM NaCl, 30 mM NaH2PO4.
2. Mix to obtain 3x PBS, pH 7.2
3. Either autoclave or filter to sterilize.
Preparation of 4% Paraformaldehyde
1. To prepare 30ml of 4% paraformaldehyde:
2. Weigh out 1.2g of paraformaldehyde
3. Add 19ml of H2O and heat to 60oC
4. In a fume hood, add 10ul of 5M NaOH. If necessary, increase the volume of NaOH added to the solution until the paraformaldehyde is dissolved [note – some particles of paraformaldehyde may not fully dissolve].
5. Add 10ml of 3x PBS.
6. Bring the pH of the solution to 7.2 by adding small aliquots (approx 5ul) of 5M HCl.
7. Make final volume to 30ml with H2O.
8. Filter solution through a 0.45micron filter.
9. Cool on ice to 4oC prior to use.
CAUTION – Paraformaldehyde is a carcinogen!
Fixation of Cells
1. Remove growth media from cells.
2. Add sufficient 4% paraformaldehyde to generously cover the cell monolayer.
3. Incubate at 4oC for approx. 30 min to complete cell fixation.
4. Cells can be stored at 4oC in paraformaldehyde for several days before use.
Using a specific fixative fluid is clearly depends on your target. There are a lot of histological books with a chapter concerning routine fixation.
Paraformaldehyde is merely the polymerized (solid) form of formaldehyde. If you dissolve paraformaldehyde in fluid, you will have a solution of formaldehyde.
The main question you need to ask is whether you should use formaldehyde prepared in your lab from paraformaldehyde, or whether you should use a commercial solution of formaldehyde, which is usually called formalin, and which contains stabilizers like methanol. This is what I would recommend; a commercial formalin solution will be a lot more stable than something you prepare in the lab. The fact that it contains a small percentage of methanol is not a problem for the large majority of procedures, and should not affect the staining that you are trying to do; methanol is in itself a mild fixative at those concentrations. It can be a greater problem to use home-made formaldehyde that doesn't actually have the concentration that you think it has, because it hasn't been properly prepared or has precipitated in part.
Indeed, Tony. I would also add that commercial solutions will be buffered at pH around 7, which is important to avoid tissue artefacts that you can get with solutions that are too acidic. This is another problem with homemade formaldehyde, the pH is not necessarily stable and you have to check before every use that it is adequately neutral.
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