fixation & decalcification - (Jul/23/2008 )

Hi,

we´re preparing tissue specimens with standard PFA fixation protocols for paraffin embedding, but we will need to add a decalcification step in between. Most protocols I found were using EDTA as a decalcifying agent. Does anyone know whether this incubation in EDTA solution should be performed in the absence of PFA, or would you recommend to use a 4% PFA solution including EDTA? I would apprechiate any comments, suggestions or protocols :-)

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Prepare EDTA solution in water and this should be fine to decalcify it.

First fix the tissue in PFA over night at RT and then next day remove the PFA and immerse the sections in EDTA solution.

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We fix overnight in PFA at 4`C and then immerse the tissue in atleast 20 volumes of 10% EDTA, pH7.3 , and take an X-ray image daily between daily buffer changes. Whole mouse hind limbs (femur,tibia& foot) with intact musculature take about 7-10 days to decal for paraffin preps.

When making the EDTA solution.... Be sure to use the tetrasodium salt of EDTA which is freely soluble in ddH2O, whereas you'll have to bring your pH up to ~9-10 to get the disodium into solution.

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