Paraormaldehyde action in immunofluorescence.

[imennajjar]

I want to know if the action of PFA (paraformaldehyde) in immunoflurescence protocoles is only fixing celles or has it a permeabilisation action.
Khanhs for Help

[Dr.Stein]

I think PFA is just a fixative. I use PFA not only for immunofuorescence but also in regular protocol preparation with standard dyes. The difference is about the dye, I think.

[imennajjar]

I ask about an eventual permeabilisation action of PFA, because we can visualise intracellular p53 (anti-P53-FITC) only by fixing by PFA 4% 20 min. So, this protocole given by one of my collegues let me uncertain. What do you think about?
Thanks a lot for your help.
Imen

[chemistry_freako]

hmm from what it seems, it does seem like it's just a fixative..

The generalized procedure for immunofluorescence labeling of adherent cell cultures involves fixation of the cells with a suitable reagent, such as paraformaldehyde, glutaraldehyde, acetone, or methanol. The organic solvents serve double duty by also removing most of the plasma membrane components, thus exposing the interior of the cell to antibodies and other non-permeant reagents. Cell cultures fixed with aldehydes require a permeabilization step with a detergent. Triton X-100 is a popular permeabilizing agent, as are saponin, Tween, and sodium dodecyl sulfate (SDS). After permeabilization, cells are treated with a primary antibody (or a mixture of two different antibodies from different hosts) followed by a fluorophore conjugated secondary antibody. In some cases, the primary antibody is labeled with a fluorophore. After immunostaining, the cells can be counterstained with other reagents to visual structural details not revealed by the fluorescent antibodies.

[Dr.Stein]

Ehehehee sorry then, I have to open my manual

[FiReaNG3L]

Yup, just a fixative. If you had used Ethanol/Methanol fixation however...

[ab06]

PFA is just a fixative. Beware that PFA may cause autofluorescence and give you a lot of unwanted background staining.

Triton X-100 is good for permeabilization. Adding some Tween 20 will help as well.

[kk]

Is it really necessary to prepare the 4% PFA/PBS fresh just before the application? It takes a long time to dissolve PFA - some labs store 4% PFA/PBS solutions at -20 C but I do not remember how their cells look like.

I prepare my PFA solution fresh: in a 60 C waterbath with occasional stiring. I add a little NaOH to facilitate PFA dissolving (5 uL 1 M NaOH to 400 mg PFA in 9 mL water - when dissolved bring it up to 10 mL by adding 1 mL 10x PBS).

Does the temperature of the PFA solution affect anything about the fixation?

After PFA fixation, I quench PFA with 25 mM glycin for 20 min. What is this last step doing exactly?

[kk]

http://publish.uwo.ca/~jkiernan/formglut.htm

A very useful link explaining details and mechanism of paraformaldehyde fixation.