Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

And Decalcified bone histology demonstrates the microarchitecture of bone showing both the mineralized and cellular components of bone, providing vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It provides beautiful images and allows for techniques such as fluro assessment and histo optometry.

Performing under calcified bone histology is technically challenging, particularly with large size specimens. This video will demonstrate the technical difficulties and methods to overcome these in obtaining under calcified sections of bone. It will not go into histo morphometric assessment of these sections.

For this, we refer you to the following important publication. This video will cover specimen preparation and fixation processing, embedding sectioning both ground and cut, slide preparation and staining following tissue collection. The specimen.

In this case, the sheep cervical spine is placed in a sealed, opaque container containing 10%phosphate buffered formula and solution. There should be minimal exposure to light if fluorochrome analysis is to be performed. As the fluorochrome are light sensitive, the specimen is then taken to the lab and trimmed to size, which improves fixation and ensures that its fits into a block.

Though orientation of the tissue on the slide needs to be known at this point so that the block can be prepared in the correct plane. Safety is very important and instruments should be used so that hands are kept free from the blade. Their area of interest is now trimmed to size.

Final trimming is then performed with a smaller band saw, which results in less tissue loss. Once a block has been cut to size, it is placed in a sealed, opaque container containing fresh formula to complete fixation. The volume of the container should be approximately 10 times the size of the specimen.

To achieve adequate fixation, the specimen should remain in the formula and solution for between one and two weeks, depending on its size, after which it is transferred to 70%Ethanol processing is then performed in a slow manner as the large size of the blocks and the low permeability of bone makes for slow diffusion of solvents. Dehydration is then carried out using ascending concentrations of ethanol and then clearing in butanol. The specimen is kept shielded from the light and under constant agitation.

We recommend approximately one week in each solution. Processing, therefore takes about four to six weeks. This should not be rushed as it will compromise the final quality of the sections.

There are a variety of embedding options available for calcified bone. The choice of resin or methacrylate depend on what is to be tested. For example, immunohistochemistry is not possible with all resins.

Epoxy resins require UV light for curing, which would interfere with fluorochrome analysis. We recommend consulting the manufacturer specifications before making a careful choice. We use TEVI 7, 100 as this allows for nice cut and ground sections for light microscopy and fluorochrome analysis and is cost-effective.

The important consideration with embedding is that the density of the bone and the density of the embedding medium should be closely matched. This is very important when later cutting the blocks, particularly for micro term sectioning, the mixture is prepared according to the manufacturer's instructions and shaken vigorously. It may be worth embedding a fuse bare samples to optimize the protocol.

Unlike with paraffin, this is an irreversible step. The blocks are placed with a cutting surface down into the mold. The mold will be bigger than the block to give support and strength.

Meth acrylate infiltration should be performed slowly to allow for maximal infiltration of the methacrylate into the bone. Again, exposure of light should be minimized. After one week an infiltration solution, the infiltration solution is changed and the hardening solution is added.

A backing plate is then fashioned. We use tevit 30 40 for this. The mold is then peeled away and the block is now ready.

Ground sectioning produces larger sections between 20 to 50 microns in thickness. It is useful for fluro analysis as thicker sections produce brighter fluorescence To produce the ground section, a microtome is needed, preferably with a diamond blade. We use a 0.6 millimeter curve diamond blade.

A lubricant is imperative. We prefer a petroleum based lubricant, although silicon based lubricants also work well. The block is then placed in the clamp.

It's secured into the macro term and the block is advanced towards the blade. The first section exposes the cutting face and orientates the section. It should be discarded.

Grinding is a slow process and should not be rushed. Adequate lubrication should be insured when the section is ground. It is then placed on a slide.

It may be cleaned and polished as required. The section has a tendency to curl, so we recommend placing a portion of a sandwich bag over it and holding it flat. With another slide clammed into place.

It is then placed into an incubator of between 60 to 80 degrees Celsius for one hour. This softens a methacrylate and helps the section adhere to the slide in a flat manner. Here we see a composite fluorescent image of three different fluorochromes chromes injected at monthly intervals.

This enables identification of the site of mineralization at a distinct time point. The doses and preparation of these labels can be found in the text. The micro term provides thin sections, which is best for light microscopy.

It is a technically challenging process. We suggest using a sledge microtone for the added strength it provides for cutting these hard blocks. A variety of blades can be used including tungsten carbide blades.

However, we recommend a stainless steel blade as this could be res, sharpened easily and frequently as required. The blade should be orientated to make a 45 degree angle with a block. The methacrylate density is susceptible to many factors, including the ambient humidity in the room.

The block should therefore be stored in a desiccate surface. Softening can be achieved by applying a wet paper to the block if required, it may take several sections to achieve the right cutting conditions to obtain a quality section. Again, the sandwich bag and additional slide are clamped to flatten the section before it is placed in the incubator.

Staining is similar to standard paraffin embedded histology except that the waxing is obviously not required. The common stains used for bone include VSA with saffron counter stain, saffron, ol light green, and mass on golden as trione. The protocol for these are referred to in the text.

The reagents are selected and filtered prior to use.Golden. The stain is frequently used for histo optometry. We recommend optimizing the stains on test slides first.

After each step, the slide can be viewed under a microscope to ensure the correct colorings. This enables achieving the correct times for each component stain. Once the stain standard optimized, all the slides from the experiment are stained simultaneously in a rack with the same protocol to avoid staining variability affecting his thermal geometry.

The slides will then cover slipped in a standard manner. We wish you luck with your experiment.