*The perfusion of animal with PBS will not do anything. It will be equal to not perfusing the animal, So PFA is a must in perfusion, in my opinion.

*

Hi Sherry

Perfusion with a buffer is done to remove blood from the vessels for cleaner immunofluorescence, especially if your structure on interest is small, like pre-synaptic vesicles. RBCs are quite autofluorescent.

I personally prefer PFA fixation via perfusion to make it quick, as post-fixation alone is super slow and cells may die before they get fixed.

In some cases, where PFA perfusion must be avoided, the brain is maintained in ice-cold buffers and sliced prior to fixation of sections.

Use of triton is not a good idea if you are interested in vesicles just under the plasma membrane like the pre-synaptic vesicles. Triton may extract cortical vesicles and you may miss your pre-synaptic proteins which will leak out.

I would recommend a very low concentration of Saponin or permeabilization by cold shock. Hope this helps

Best

Aparajita

Hi,

The Perfusion is done to have quicker fixation so if you studied sensitive structure or ultrastructure sometime is better to do PFA perfusion

I did not work with brain tissue, but I did cochlea synapse labeling and long PFA fixation can reduce the labeling

For permeabilization and blocking I performed 30min incubation with this solution: 30% serum, 0.3% Triton 100X, PBS1X. And I obtained pretty good Synapse staining

If you think triton is to agressive for you structure you can try Tween solution

Hope this helps

Best

Corentin