All histological procedures can be divided into a similar series of steps.
For the paraffin method these steps are as follows:
1 Tissue resection
2 Fixation
3 Dehydration
4 Infiltration and embedding in paraffin
5 Sectioning with a microtome
6 Mounting on microscope slides
7 Clearing and Staining
8 Preparation of permanent mounts
1,2,3-Tissue resection, fixation and dehydration.
Follow common techniques as adopted by most small laboratories performing this procedure, but not covered in this blog.
4-Infiltration and embedding in paraffin.
The tissue blocks must be infiltrated with paraffin wax to provide structural support during the sectioning process. Paraffin wax dispensers aid this process by keeping the wax at optimal temperature throughout the procedure. During infiltration the wax will equilibrate in the tissue block, and occupy every space it can find, originally occupied by the fixative solution. After infiltration the tissue is allowed to solidify in a mould, embedded within a small cube of paraffin.
5-Sectioning.
Sectioning is carried out using a microtome – the microtome will draw a knife sharp blade across the paraffin block and produce a series of thin sections of uniform thickness. The objective is to make a ribbon of sections of uniform thickness for optimal viewing later.
6-Mounting on a microscope slide.
Each section can be mounted on an individual slide by using a paraffin section mounting bath, which allows the warm water held within to ‘float’ each section onto a slide. The wax ribbon will be slightly crinkled as they are cut and the bath helps to flatten this out. Its important to do this slide by slide from the ribbon, so that the slices can be chronologically identified. The bath should be kept at 45 degrees C throughout the mounting stage.
A slide drying bench can be used so that the Microscope slides dry out uniformly and are kept in order.
7- Clearing and staining.
Before the slide tissue can be stained the wax needs to be cleared from the section- ‘Clearene’ is a product designed to replace xylene and toluene for clearing sections. There are many stains available depending on the structures of interest in the tissue section.
The two most commonly used stains are Haematoxylin and Eosin Y. Haematoxylin stains negatively charged structures whilst Eosin Y will colour the remaining cell structures. If this dye is to be permanent then the dye must be oxidised with Scott’s Solution.
REAGENTS: Scott’s Tap Water/Bluing – Magnesium sulphate (MgSO4)30.0 gram Sodium bicarbonate 2.0 gram Tap water 3000.0 ml
8-Mounting
Once staining is complete the tissue samples need mounting with a proprietary mounting resin and a cover slip. Once examined slides should be stored in purpose made storage boxes.
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