Re: how are bones prepaid for histological studies?

Dear Mike,

My laboratory performs histological studies on bones all the time, mainly 
on rat and mouse bones, in which we study factors that affect bone growth 
and metabolism. Exactly how we prepare it depends on what we want to look 
for. There are whole books on this subject, but I will give you an 
overview of two different methods that we use most often. One of the main 
issues is dealing with hardness of the bone mineral so it won’t be a 
problem when cutting sections from the tissue. There are two approaches. 
Dissolve it out of the bone, or use a very hard plastic to embed it.

The most generally useful method is regular formaldehyde fixation and 
paraffin embedment. For very highly detailed histology or even electron 
microscopy, we use glutaraldehyde fixation and embedment in hard epoxy 
called (Epon). And there are other ways as well.

To get good fixation, we dissect the bones and usually split them down the 
middle to get good fixation inside as well as outside. Then we immediately 
place them in an ice cold solution of 4% formaldehyde in a neutral buffer 
and fix them overnight. You can do the same thing with human bone biopsy 
samples. 

Then we rinse the fixative away with cold saline. To use paraffin, which 
is by far the most common histological technique, the mineral has to be 
removed, or when you try to cut sections, the whole block rips apart. To 
get rid of the mineral, we store the samples in the cold in a 10% solution 
of EDTA buffered to pH 7 with Tris-HCl. The EDTA extracts the mineral from 
the bone and leaves it flexible. This could be done with any acid, but 
EDTA is a gentle way to do it and not damage the tissue. How long this 
takes depends on the size of the bone. Very small samples may take only a 
couple of days, but larger samples may take a couple of weeks. The 
solution should is changed every couple of days. After the bones are de-
mineralized, they can be treated like other tissue samples, and processed 
for paraffin embedment. This entails dehydration with a graded series of 
ethanol solutions, then xylene, then putting them in heated paraffin, and 
letting the block cool. After that they are sectioned using a histological 
microtome.

For Epon embedment, the trade-off is much higher resolution, but samller 
tissue block size, since they must be cut on an ultramicrotome. The bones 
are split as above and then fixed for 2 to 4 hours in cold, 2.5 % 
glutaraldehyde in a buffer that contains 0.1 M sodium cacodylate, pH 7.4. 
This is removed by 3 rinses in cacodylate buffer with 7% sucrose, then 
just cacodylate buffer overnight. Because the plastic embedment material 
is about as hard as the bone, these samples don’t strictly require de-
mineralization. But it can give better detail in the sections if they are 
demineralized, using EDTA as above. The embedment is the same as for 
electron microscopy and requires dehydration, infiltration with the 
plastic resin, and slow baking to harden the plastic. After that, sections 
can be cut using a glass knife on an ultramicrotome for light microscopy – 
typically around 1 micrometer think. For electron microscopy, thinner 
sections must be cut.

Advantages to paraffin: done routinely in most histology labs, bigger 
tissue block size, so you can see more the bone in one section. It also 
can be used for many staining procedures, including RNA localization (in 
situ hybridization), antibody staining, enzyme activity (many enzymes are 
still functional after processing), and other methods. Disadvantages: less 
histological detail is preserved, and the thicker sections (usually 5 to 
10 micrometers thick) can also contribute to lower resolution. 

Advantages to Epon: extremely high resolution, and cellular details are 
preserved beautifully. Can be used for electron microscopy, too, and can 
also be used in studies where the mineral needs to be in the tissue. 
Disavantages: more time-consuming, smaller block size, requires special 
materials and equipment, and doesn’t allow probing with antibodies – 
except after some special procedures (etching with alkali in ethanol) to 
remove the plastic, and even then the ability of antibodies to react with 
the tissue is often lost. 

So there are two of the most common methods. There are many, many more. 
There is a recent book, Handbook of Histology Methods for Bone and 
Cartilage, edited by Y. H. An and K. L. Martin, published by Humana Press, 
Totowa, New Jersey (USA), 2003. This book will undoubtedly give you more 
than you ever wanted to know.

Paul Odgren, Ph.D.
Dept. of Cell Biology
University of Massachusetts Medical School
Worcester