For how long should rat limb be kept in 4%PFA @4C for fixation prior to decalcification?

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Janine M. Ziermann

24 hours at 4C is sufficient

James L Borke

Over night if the skin is removed.

Vehid Salih

What is key is any fixation method is the volume of fixative relative to the volume of tissue sample. In normal routine histology utilising formalin, 10-20% is recommended depending on the tissue being fixed (i.e. it's density viz: brain vs lung vs liver vs muscel etc). 4% PFA is a much more 'gentle' fixative compared with formalin.

It also depends on how the tissue will be analysed i.e. whether it is for fluorescence imaging, routine histology, or special stains.

As it is a decalcification method to be used subsequent to fixation, then tissue morphology is paramount - I would like to suggest 36-48 hours at 4C.