Paraffin wax is the most commonly used medium for embedding tissue samples prior to staining and sectioning, particularly for the brain which would otherwise be too fragile for micro-thin sections to be prepared for microscopic analysis.

Fixation is first employed prior to embedding in order to preserve the cellular architecture and composition by halting further biochemical reactions as well as strengthening the tissue which increases its resilience to damage by further processing.

Perfusion is the preferred choice for fixation as it has the most rapid tissue penetration, utilizing the cardiovascular system of the live anaesthetized mouse to infuse the tissues with fixative (Kalimo, 1976).

Immersion fixation is an alternate method, whereby the mouse is dissected to harvest its brain which is immersed in a fixing solution, a slower process which relies on the rate of diffusion to penetrate the tissue completely and is influenced by pH and temperature. A greater level of autolysis and putrification can occur with immersion fixation than perfusion due to the slower rate of tissue penetration of the fixative. 10% Formalin penetrates tissue at a rate of about 1mm per hour so the brain should be immersed in formalin for at least 24 hours if non-perfused.

To fix and process the mouse brain, it is immersed to sequentially replace the fixing solution with intermediary solutions and ultimately molten paraffin wax. The first step is a phosphate buffered saline solution wash which is repeated three times to remove fixing solution. This is followed by dehydration with alcohol to remove water as paraffin wax is hydrophobic. The mouse brain is inserted into a plastic perforated tissue cassette which serves as a protective cage during processing and allows the specimen to be labelled. The next steps are ideally carried out in a processing machine such as a tissue-transfer which automates the sequential immersion into different graded solutions which become progressively more hydrophobic. Or a fluid-transfer machine where the specimen sits in one chamber and the different solutions are successively pumped in and out. The processing can also be carried out manually but this is rather laborious and the automation machines can process multiple specimens rapidly, simultaneously and consistently. Automation machines would typically immerse the tissue in a dozen or so solutions which get progressively more hydrophobic. The concentrations of solutions and length of time in each immersion may vary. The process takes about 16 hours, after which the mouse brain impregnation with paraffin wax is complete.

For automation methods, the plastic cassette containing the brain specimen is placed into a stainless-steel processing basking and dehydrated by immersing sequentially into graded alcohol solutions of 70%, 90% and finally 100% ethanol.

The next stage is clearing to displace the ethanol from the tissue with solution that is miscible with paraffin wax. The mouse brain is immersed in three different solutions of 100% xylene or histo-clear: a less toxic alternative. These are suitable for sequential displacement of the solution infiltrated into the brain tissue as both are miscible with alcohol and paraffin wax.

The infiltrate the mouse brain with paraffin wax, the wax is heated to approximately 60°C where it is molten and the brain is immersed into molten paraffin wax several times to ensure the prior infiltration solution has been displaced and the wax has fully impregnated the tissue.

The specimen holding cassette is then transferred to a holdall bath containing molten paraffin wax whilst the embedding station is prepared to embed the mouse brain. The brain is removed from the plastic cassette and placed in a metal base mould, prewarmed to ensure the wax does not rapidly solidify. The brain is aligned in the mould optimally for sectioning. Molten wax is then poured to fill to the top of the mould, thoroughly submerging the mouse brain. The labelled plastic cassette base is immediately placed on top of the metal base mould which is then placed onto a cold plate to accelerate solidifying of the wax. The wax block is removed from the mould which may require a sharp scalpel to ease the wax block away from the mould. The plastic cassette base forms the base of the wax block and can be clamped in a microtome for sectioning.

The mouse brain is now embedded into paraffin wax block which can be stored at 4°C for future use, or immediately sectioned using a microtome. By embedding a mouse brain in paraffin wax, the brain tissue is imbued with the wax and adopts its firm consistency allowing for sections 4-5µm to be cut using a microtome with minimal compression occurring, thus conserving fine cellular structure (Gunasegaran, 2007).

 

Gunasegaran, J.P., (2007) Textbook of Histology and Practical guide.

Kalimo, H., (1976). The role of the blood-brain barrier in perfusion fixation of the brain for electron microscopy. The Histochemical Journal January 1976, Volume 8, Issue 1, pp 1–12