> while I am well fixed tissue (rat-pup brain) for
> vibratome sectioning, 4% PFA fixation for
> immunohistochemistry is yielding poorly fixed tissue
> even with over night fixation.
>> Could I
> a. use 4% PFA in PBS instead of 4% PFA in
> phosphate bufer.
> b. increase the concentration of PFA as the
> powder is quite old.
4 % PFA in PBS has worked fine for me. Wash the cells in cold PBS twice,
add 4 % PFA in PBS and let stand at room temperature for 30 minutes.
Pour off the PBS containing 4 % PFA, and add fresh cold PBS. Store at 4
(hint: by not washing but just changing the media after PFA fixation, you'll
use the leftover PFA to avoid bacterial / fungal growth)