I want to prepare fixation buffer 4% PFA/PBS but I'm confused with the methods I've come across.
I tried to follow this one method whereby I dissolved 4g of PFA (merck, for synthesis) in 50 ml of DPBS 1X (Gibco, no Ca, no Mg) on hotplate with temperature of about 60 degree, but it didn't dissolve completely and there were white sediments at the bottom. I still tried to filter the solution with a filter paper and managed to get 40ml of clear solution.
Now, is this okay and can I use this to fix my embryos.... or must I make a new one and dissolve 4g of PFA in 100ml 1X PBS instead, like how this one method instructs? or is there another way to prepare it...?
Here goes, my protocol for making 4%PFA (100ml).
1. Take 80 mL of 1X PBS to a glass beaker on a stir plate in a ventilated hood. Heat while stirring to approximately 60 °C. Take care that the solution does not boil. Then Add 4 g of paraformaldehyde powder to the heated PBS solution.The powder will not immediately dissolve into solution. Slowly raise the pH by adding 1 N NaOH drop wise from a pipette until the solution clears.
2. Once the paraformaldehyde is dissolved, the solution should be cooled and filtered.
Adjust the volume of the solution to 100ml with 1X PBS.
3. Recheck the pH, and adjust it with small amounts of dilute HCl to approximately 6.9.
The solution can be aliquoted and frozen or stored at 2-8 °C for weeks or -20°C for months.
Just use 37 % stock formalin available anywhere to prepare 4% solution in PBS , you do not have to dissolve anything, whenever it says parafirmaldehyde just use formalin. In case you can not have methanol,
The pH is critical, see e.g.
https://www.rndsystems.com/resources/protocols/protocol-making-4-formaldehyde-solution-pbs
And answers to the same question on RG:
https://www.researchgate.net/post/How_to_make_up_paraformaldehyde
Here goes, my protocol for making 4%PFA (100ml).
1. Take 80 mL of 1X PBS to a glass beaker on a stir plate in a ventilated hood. Heat while stirring to approximately 60 °C. Take care that the solution does not boil. Then Add 4 g of paraformaldehyde powder to the heated PBS solution.The powder will not immediately dissolve into solution. Slowly raise the pH by adding 1 N NaOH drop wise from a pipette until the solution clears.
2. Once the paraformaldehyde is dissolved, the solution should be cooled and filtered.
Adjust the volume of the solution to 100ml with 1X PBS.
3. Recheck the pH, and adjust it with small amounts of dilute HCl to approximately 6.9.
The solution can be aliquoted and frozen or stored at 2-8 °C for weeks or -20°C for months.
Agree with the following: Slowly adding 1 N NaOH drop from a pipette until the solution clears.
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