Dear Julieta G García-Donas

The following 2 methods are good for bone preparation

Methods and Protocols for Decalcification of Bone Material

http://www.ihcworld.com/_protocols/histology/decalcification.htm

Paraffin Processing of Tissue:

http://www.protocolsonline.com/histology/sample-preparation/paraffin-processing-of-tissue/

http://www.ihcworld.com/_protocols/histology/decalcification.htm

http://www.protocolsonline.com/histology/sample-preparation/paraffin-processing-of-tissue/

Dear Hesham N. Mustafa

Thank you so much for the links.

I have previously prepared dry bone for histological analysis (ribs) (following e.g.  Paine (2007) Do it yourself. Journal of Anthropological Sciences. Vol. 85 (2007), pp. 213-219) and I did not experience any problem. But with the femora, I have some samples that crashed (I mean the mounting slide gets broken like if it would have had an impact on the middle and ends up breaking). I was wondering if the SIZE of the bone may have something to do or maybe the quality of the slide....Did it happen to you?

Dear Julieta G García-Donas

I think your problem is related to the decalcification period that must be shortened depending on a pilot study.

The issue is not the size of the bone but the efficiency of the microtome used that will provide a good quality of the slide.

I suggest to repeat again with the cautions of decalcification.

Hello Julieta.  You have not clearly explained to Heshem that you wish to keep the mineral intact, that you have no desire to decalcify the bone.  By keeping the mineral, you can perform investigations that relate to variability in mineralization density, and other studies that would be impossible with decalcified bone.

All of the techniques for embedding, cutting, grinding, and polishing histological thin sections of femur bones are well understood.  We specialize in this, cutting sections from adult humans and gorillas with 100% success; we have not had a failure in many years.  In this case, the method described by Paine would be performed only in the case that you had to keep costs down, or when you do not have access to a suitably equipped laboratory.  But quality control when following this method will be difficult, and that is what causes the problems you are having.  

There are some very fine laboratories in the UK, such as Alan Boyde's at Queen Mary Uni Lond, but also probably at York, Liverpool, and a few other places.  Without actually observing your setup and procedures, I can advise a couple of things that may help.  First is to use "plexiglass" plastic slides.  They are non-flexible slides made from polymethylmethacrylate, that are 1.5 mm in thickness and more, which are very stiff and will not break.  Those made by Exakt are probably the best, though a little expensive (this is NOT the Exakt power saw company).  I don't know where you can find them in the UK, but here is a site you can order large slides from:

http://histolab.e-line.nu/en1/nya_e-line/Instruments/Accessories_for_Instruments/Plexiglas_50x100x1,5mm_25_st_fp_Exakt?id=EX-41510

Exakt also makes a 50x76 mm slide, which is a good size for human femurs, but I do not see that size on this site. 

In addition, the ground and polished block should be mounted onto the slide with cyanoacrylate glue, sometimes called "super glue".  But you do not want the kind you can get at the hardware store.  You need to go to a chemical supplier and request "3M™ Scotch-Weld™ Instant Adhesive CA7",  The CA7 formula has a specific viscosity that makes it work well.  It is expensive, but you don't need much, and one 1 oz bottle will mount a hundred blocks.

Dear Prof. Bromage,

Thank you very much for your message. The laboratory that we have it is fully equipped (we have Buehler equipment) for histological analysis so the processes of cutting, grinding, polishing....follow the standard procedures for histological preparation of bone. It is true that with Paine´s method we have experienced similar (drawbacks as the one I am explaining in this conversation. It is the first time I process femora (I have been working with ribs) and I was just wondering that the problem I have was the one you have suggested in your message. The thickness of the slides that I am using cannot support the size of the bone....and therefore, they break. I will ask the University to order the glass slides you suggested. I will let you know if I successfully prepare the sample.

Thanks again

Glad to hear that you have a well equipped lab.  But to be clear, the slides are 1.5 mm plastic (PMMA).  Let us know if you would like a complete protocol.  

Thanks for the clarification about the size of the slides.

Definitely, Would you mind to send me a complete protocol? It sound reasonable to have a look and compare the one that I am using now and yours and see what I can improve.

Thanks again!