When preparing a sample (or multiple samples) for histology microscopy, there are multiple steps required. We’ve covered these steps in brief in a previous article on How Histology Slides are Prepared, but this article will focus on one particular procedure which needs to take place between tissue fixation and the embedding/sectioning of paraffin blocks: tissue processing. You simply can’t take fixed tissue and embed it! We have already introduced fixation in this article and embedding/sectioning in this article.
Following fixation, tissue is transferred to a tissue cassette- see the multicolored examples below!
Get your pencil out
These come in various sizes and hold and protect the tissue whilst it undergoes processing. Once the embedding stage is reached, the cassette lid is snapped off and the main part of the cassette forms a base for the paraffin wax block. The cassettes can be labelled by hand (with pencil!) or your histology lab may have a cassette labelling machine.
There are three main steps in tissue processing, namely: ‘dehydration’, ‘clearing’ and ‘infiltration’. Each of the steps of the processing method involves the diffusion of a solution into tissue and dispersion of the previous solution in the series.
All the fun of the carousel
In most modern institutes and histology labs, processing will be carried out in dedicated tissue processing machines. The older design of machine is a carousel which contains a cage in which the tissue cassettes are placed. This carousel has a number of glass beakers containing solvents and solutions which ensure that the tissue is dehydrated and cleared ready for paraffin wax embedding. The carousel vertically agitates the cage in each solution before moving on to the next solution in the dehydration/clearing method.
The modern processors have a chamber in which the specimens are held and the different solutions are pumped in and out of the chamber. In general, the whole process takes around six hours and is usually set up to run overnight.
First, remove the water
Firstly the tissue needs to be dehydrated to remove the water from the tissue which is present- either bound to the tissue, or free in the tissue. Paraffin wax is hydrophobic, therefore, most of the water in the tissue must be removed before it can be infiltrated with wax. This process is carried out by immersing tissue in a series of ethanol solutions of increasing concentrations until 100%, water-free alcohol is reached. A series of increasing concentrations is used to ensure that the water in the tissue is gradually replaced by the alcohol and to avoid excessive distortion of the tissue.
Various components of the cell are also removed by this process. At the lower end of the ethanol concentrations, water soluble proteins are removed, whilst towards the 100% ethanol step, certain lipids may be dissolved.
Ethanol and wax don’t mix
Although the tissue reaches the final stage of dehydration in 100% ethanol, it’s not possible to proceed straight to wax embedding- ethanol and wax don’t mix!
This is where ‘clearing’ comes in. The term ‘clearing’ refers to the property of the solvents used- they have a relatively high refractive index and when tissue is immersed in it, it becomes transparent and clear.
It’s becoming clearer…
The solvent used for this intermediate stage is usually xylene. The ‘clearing agent’ needs to be miscible with both ethanol and paraffin wax. Following the dehydration, the tissue is immersed in one to three different xylene immersions. In these stages, the ethanol is gradually replaced with xylene and when the tissue is embedded, the xylene will be replaced by the molten paraffin wax.
Shrinkage of tissue can occur at these final stages as the xylene also removes fat residues left in the samples.
At last – a block!
The final stages are called ‘infiltration’ and ‘blocking out’. Infiltration is when the final xylene is replaced with molten wax which infiltrates the tissue. Again, this is typically three different wax immersions to ensure that none of the clearing agent remains in the tissue. After the final infiltration, the tissue cassettes are transferred to an embedding station. This machine has reservoirs of molten wax, hotplates and a cold plate for setting the blocks. The infiltrated tissue is removed from the cassette and orientated within a suitably sized metal mould. The mould is filled with molten wax, the main part of the labelled cassette is placed on top and this is also filled with wax. The whole mould is transferred to the cold plate to finally set.
That ends the journey from tissue to wax block, which is, I guess, the start of another journey of sectioning, making slides and immunohistochemistry!
Finally, below is a table which highlights the typical main stages of tissue processing;
|Dehydration||70% alcohol||60 mins|
|Dehydration||90% alcohol||45 mins|
|Dehydration||Absolute alcohol||45 mins|
|Dehydration||Absolute alcohol||45 mins|
|Dehydration||Absolute alcohol||60 mins|
|Infiltration||Paraffin Wax||30 mins|
|Infiltration||Paraffin Wax||60 mins|
|Infiltration||Paraffin Wax||90 mins|
|Blocking Out||Paraffin Wax||n/a|