Fixation is the most important step in the histological preparation process. Its purpose is to preserve structures and stabilise cellular material against the potentially damaging effects of certain analytical techniques such as staining and immunohistochemistry.

The fixation time depends on the size of the sample. At least 10 volumes of fixative are used in relation to the size of the tissue. In our case the fixation time ranged from 48 hours to several days.

Samples are fixed in 10% volume formalin for 24h 0m 0s to 168h 0m 0s at 4°C

Samples are prepared differently, depending on the type of sample and what you want to highlight.

For teeth, a cross section is required to measure the depth of each component of the tooth.

For a bovine bone fragment (humerus, femur, incisor) measuring 2 cm x 2 cm, the following conditions were used

Soften the tissue by decalcifying it

Place samples in a solution of 10% volume EDTA in 1x PBS (pH 8) for 4h 0m 0s

and 0h 30m 0s at 50°C in a KOS microwave tissue processor (Milestone Medical)

Samples should be placed in specially designed cassettes to hold and flatten them. For very small samples (less than 2 mm) plastic foams are available to hold the sample in place

Cassettes should be marked with a pencil (water and alcohol resistant) or a cassette printer

Record as many details as possible: date, project name, individual number, tissue nature, type of section

Dehydration replaces the water (80%) in the tissue with ethanol.

To do this, the cassettes are placed in an automatic dehydration machine which dehydrates the tissue using different alcohol solutions of increasing concentration (70% to 100%)

The machine then clarifies the samples with 2 xylene baths, removing the ethanol as paraffin is immiscible in alcohol

Finally, 3 paraffin baths at 60°C (liquid) are used to impregnate the tissue. Finally, the samples are immersed in several paraffin baths.

Each step takes between 2 and 4 hours, depending on the size of the samples. For our samples, we left the fragments for 4 hours in each bath.

A	B	C
Steps	Bath	Time (h)
1	70°C ethanol	4
2	80°C ethanol	4
3	95°C ethanol	4
4	100°C ethanol	4
5	Xylene	4
6	Xylene	4
7	Paraffin 1	4
8	Paraffin 2	4
9	Paraffin 3	4
	Total	36

Tissue dehydration, clarification and impregnation baths

Embedding allows the specimen to be oriented and fixed with a paraffin embedding medium so that it can be sectioned later

Place the specimen in the bottom of a metal mould

Place the appropriate cassette on the mould to allow identification of the tissue embedded in the paraffin block

Once the mould is filled, position the tissue

Allow the block to solidify

Wait approximately 15 minutes in the refrigerated zone until the block has stiffened and can be removed from the mould

This step can be performed using an embedding station

Cut the blocks into trapezoidal shapes to make it easier to cut strips to prepare the slides

Surface of the paraffin blocks to reveal the tissue. Use a microtome set for a 15 micron cut

Slightly rehydrate the surfaced samples by placing them on damp paper at 4°C. This will reduce the brittleness of the tissues and make them easier to cut

The samples are sectioned using an automatically rotating microtome (Histocore autocut). This is an important stage in the preparation of slides as it determines how well the tissue can be observed under the microscope

Make ribbons of cuts:

• Set the cut size to 5 microns
• Position the block, with the smaller side of the trapezoid facing up, as close as possible to the blade using the slider
• Unlock the blade guard
• Turn the crank clockwise
• Gently pull the ribbon with a brush to help it unwind and to avoid creasing the tissue

The paraffin ribbons are placed in a water bath for a few seconds to allow the heat to unfold them

The ribbon is then ready to be placed on a glass slide

The use of adhesive slides is recommended

The slide is then dried on a slide holder in a laboratory at 40°C for at least

Slides, that have not been stained at this stage, are called "white" slides

Before staining, the paraffin must be removed to allow the dyes to penetrate the tissue. This step is usually included in the automated steps of a staining robot

Haematoxylin, eosin (HE) is a morphological usual stain. Haematoxylin is a basic dye that stains acidic structures a purplish blue, such as DNA in nuclei. Eosin is anionic and acts as an acid stain. It will therefore stain basic structures such as cytoplasm pink

The slides are then automatically scanned and digitised using the Pannoramic SCAN/3DHistech tool

They can then be viewed from any computer using the dedicated CaseViewer software (freeware). The quality of the scanner allows colour sections to be magnified up to 63 times