Paraffin Embedding

Five major steps are involved in paraffin embedding: fixation, dehydration, transparentizing, immersion and embedding.

Fixation

Please refer to the Fixation section described above.

  • Fixation
    Please refer to the Fixation section described above.
  • Dehydration
    This step removes water completely, creates a condition for the next step and hardens the tissue of interest. The dehydrating agents describes below are completely miscible with water and can be prepared in different volumetric ratios with water.
    • Ethanol
      As the most commonly used dehydrating agent, ethanol has a strong water separation and tissue hardening capability. However, since ethanol has strong penetration and contractility, its concentration should be progressively increased to avoid tissue excessive shrinking.
    • Acetone
      As a usual substitute for alcohol, acetone acts as both a fixing solution and fast dehydrating agent. Pay attention to the dehydrating time with acetone as it tends to over harden tissues.
  • Transparentizing
    After dehydration, the tissue of interest requires a transparentizing step because the dehydrating agent used in the previous step is immiscible with the paraffin from one of the subsequent steps. The addition of transparent reagent helps paraffin absorb into the tissue. Common transparent reagents are:
  • Xylene
    As the most widely used transparent reagent, xylene is miscible with both ethanol and acetone, and it acts as a fusing agent for paraffin wax. Since xylene has a strong and fast contractility to tissue, the tissue should not be immersed for an extended time period or it will be over crisp and too hard.
  • Benzene and Toluene
    These reagents are similar to xylene. However, they have weak and slow contractility to tissue, and therefore the tissue can be immersed in these reagents for a longer time. Note that benzene and toluene have high toxicity and must be handled with care.
  • Chloroform
    Compared to xylene, benzene and toluene, chloroform is a much gentler reagent. However, it has a small refractive index, and the tissue should thus be immersed in chloroform for a longer time than the other transparent agents in order to guarantee complete penetration.
  • Cedar Oil
    Due to minimal sclerification created by cedar oil, it is an appropriate transparent agent for fine and soft tissues. Super hard tissues (e.g. skin tissue) and dense fibrous tissue are also easier to be sectioned after immersing in cedar oil. However, this oil is not useful for other common tissue sections because of its high concentration and weak penetrability.

In recent years, a new type of environment-friendly transparent agent has appeared in market. Instead of aromatic compound, the main components of this new reagent is alkanes, and it can be used to replace xylene.

  • Immersion
    After transparentizing, the tissue can be immersed in molten paraffin wax so that it adsorbs the wax-substituting transparent agent. Based upon the melting point of wax, immersion should be performed at 54-64℃.
  • Embedding
    This is a process of treating the tissue in a paraffin box so that the paraffin wax cools down and solidifies. The treatment conditions (using ethanol and xylene as an example) are shown in the table below. After cooling is completed, the tissue will be ready for sectioning and suitable for storage.
Step Reagent Time (Hours)
1 75% Ethanol 0.5 to 2
2 85% Ethanol 0.5 to 2
3 95% Ethanol 2
4 95% Ethanol 2
5 95% Ethanol 2
6 100% Ethanol 0.5 to 1
7 100% Ethanol 0.5 to 1
8 100% Ethanol 0.5 to 1
9 Xylene 0.25
10 Xylene 0.25
11 Xylene 0.25
12 Paraffin Wax 0.5
13 Paraffin Wax 1 to 2
14 Paraffin Wax 1 to 2

Tissue Section Types

  • Frozen
    The most important feature for this type of tissue section is to keep antigen’s immune-competence completely, especially for the cell surface antigen. Both fresh and fixed tissues can be processed as frozen tissues. However, the tissues must be dried (or primary fixed) and stored at low temperature.
  • Paraffin-Embedded
    Paraffin-embedded tissue section is normally sliced by a rotary microtome to give a thickness of 2-7 μm. With proper treatment, the section reveals clear tissue structure and exact antigen location to enable high medical-value pathology researches and retrospective studies. This section type can be stored at 4℃ for long term use.

Processing

  • Cell on Coverslip
    • Place settled coverslip in culture bottle or perforated plate
    • Take out coverslip after cell growth has reached 60%
    • Fix coverslip with cold acetone or 4% paraformaldehyde for 10 to 30 min
    • Store cell (in gelatin) at -20℃
  • Cell Smear
    • Collect non-adherent cells and wash 2X by cold PBS buffer
    • Re-suspend cells with PBS buffer
    • Add 30-50 µL to settled slide and smear it evenly
    • Air dry slide a little bit and cover cell with 4% paraformaldehyde for 2-4 hours
    • Store cell (in gelatin) at -20℃