You could try decalcifying the bones at 37 degrees and increasing the EDTA concentration. We decalcify mouse and rat heads at 37 degrees for 2 weeks in 25% EDTA.

I agree with Nathan that some of your tibias are not fully decalcified. What to do depends on what you intend to do with the sections. If you only wish to use histochemical staining methods - prolonged decalcification is an option, but some of the methods may need to be modified with longer exposure to the dyes.

If you intend to do immunohistochemistry, the longer decalcification is often detrimental for good staining (in my experience and documented in the literature), but it differs for different targets for the antibodies - and some work well (e.g. Abs to matrix components).

For histology methods, Bancroft Histological Techniques recommend re-embedding the block after at lest overnight (up to a week) bath in molten wax. One of the best approaches is double-embedding (combining nitrocellulose and paraffin as embedding media) which is especially useful for tissues of mixed consistency - as in your case with healthy bone and tumour infiltrate). I have diagnosed human bone material prepared in this way and the histological detail in large sections was exceptionally good - you could try long impregnation with wax (worked for me for calcified arteries) and double embedding in case it doesn't work well.

Ideally, if you intend to do IHC, the whole material should be processed in the same way, which means that it should be reversed to decalcification stage (at room temperature or at 37 deg C), but if you are in the middle of cutting the processed material - dealing with blocks which 'crumble' is your best option.

If you have blocks which 'crunch' when cutting, but you do not have large parts falling out - you could try surface decalcifying - it works for 1-3 sections, even if the tissue is in wax - hard to explain, but an 'old school' technician preparing sections for me showed me some good results.

Advice for cutting - increasing tilt of the knife also helps.

Good luck with your project.

Hi Mackenzie,

You can try following steps:

1. Use higher EDTA concentration (15% at 4℃ for 2 weeks with change every alternate day) to make the tissue soft enough;

2. Trim unnecessary part of the bone to make it smaller size before decalcification;

3. Use sharp blade for sectioning.

Hope it will help to fix your problem.

All the best.......