HISTOLOGY LABORATORY HANDOUTS
Histology & General Histological Techniques II
    Dehydration, Clearing, Infiltration and Embedding
      See also other Histological Techniques
        Animal Dissection and Fixation of Tissues
        Tissue Processing: Sectioning And Slide Mounting
        Stains and Staining

    A. GENERAL PARAFFIN PROCESSING ROUTINE

    I. Summary
    Tissues need to be processed through solvents with decreasing water concentrations inorder to infiltrate them with paraplast. You will transfer your tissues through increasing alcoholic solutions. They will then be "cleared". The clearing agent is soluble in both alcohol and paraplast. The will then be infiltrated with Paraplast, blocked and stored until sectioning.

    Dehydration Methods:
    Several different methods are available. Alcohol is the most common solvent used for both dehydration and rehydration. Dioxane is also a clearing agent. It provides for a more rapid processing of tissue and functions as a clearing agent, but is also toxic. See details below under clearing agents.

    Alcohol Protocol
    Alcohol solution
    95% Alcohol
    95% Alcohol
    100% Alcohol
    100% Alcohol
    Time
    .5 to 1 hours
    .5 to 1 hours
    .5 to 1 hours
    .5 to 1 hours
    Dioxane Protocol
    -- In tissue rotator underhood
    Dioxane solution
    Dioxane I
    Dioxane II
    Dioxane III
    Time
    15 minutes
    15 minutes
    15 minutes

    Clearing Method:
    Clearing solution
    Alcohol /Xylol 1:1
    Xylol I
    Xylol II
    Xylol /Paraplast 1:1
    Time
    15 minutes
    15 minutes
    15 minutes
    15 minutes
    Infiltration:No Longer Than 4 Hours
    Paraplast changes
    35o Celsius
    Paraplast I
    Paraplast II
    Paraplast III
    Time
    30 minutes
    30 minutes
    60 minutes

    II. Protocol

      We will use the following schedule to process our tissue. No Times are listed. Please fill in

      Start __________________________ and finish during Laboratory in the afternoon

      Dehydration Clearing Infiltration:
      Change to Time Change to Time Change to Time
      95% Alcohol
      95% Alcohol
      100% Alcohol
      100% Alcohol
      _________
      _________
      _________
      _________
      Alcohol /Xylol 1:1
      Xylol I
      Xylol II
      Xylol /Paraplast 1:1*
      _________
      _________
      _________
      _________
      Paraplast I*
      Paraplast II*
      Paraplast III*
      _________
      _________
      _________
      Oven temperature at * 35o or the lowest remperature for the melting point of the paraplast being used. Note: the higher the melting point the harder the paraplast. The higher the melting point the greater possibility you may "cook" your tissue!!

      Embedding at _____________________ PM

      Embedding: Will be Demonstrated

      1. Spray a stainless steel mold with releasing compound
      2. Pour a small amount melted Paraplast (fresh) into it.
      3. Transfer the tissue with hot forceps and orient it properly in the center of the depression.
      4. Place the white plastic form on top of the mold and fill with melted paraplast.
      5. Remove from heat. Cool the top surface of the Paraplast by blowing gently on it.
      6. As soon as a scum of Paraplast has formed on top, sink the cup gently into a cold water bath.
      7. Cool thoroughly in cold running tap water.

    B. GENERAL PARAFFIN PROCESSING INFORMATION

      A. General Dehydrating Protocol
      A standard graded series of alcohols are used most frequently to dehydrate or hydrate tissues to prevent shrinkage or distortion of the cells. This is used when one immiscible solvent must be replaced by another. The following represents standard dehydration procedures.

      Dehydration
      in Preparation for Embedding
      Dehydration After Staining Slides
      in Preparation for Coversliping
      Aqueous fixative
      Water
      35% Ethanol
      50% Ethanol
      70% Ethanol Storage
      70% Ethanol + Lithium carbonate
      treatment for picric acid
      95% Ethanol from Alcoholic fixative
      95% Ethanol (+0.5% eosin for tiny,
      transparent objects.)
      100% Ethanol
      100% Ethanol
      CLEARING Paraffin or other
      Embedding medium
      Aqueous stain
      Water (rinse)
      35% Ethanol
      50% Ethanol
      70% Ethanol Storage
      95% Ethanol
      95% Ethanol
      (+ eosin for counter stain if used.)
      100% Ethanol
      100% Ethanol
      CLEARING resin medium

      For Tissue preparation one to two hours in each solution should be adequate. Tissues with a high water content such as embryo tissue would require a much shorter time. To ensure complete removal of water during dehydration, use two changes of 100% ethanol of at least one half hour each. Never leave tissues in 95 or 100% ethanol more than a total of 2 hours or the tissues will harden. Tissues can be stored in 70% ethanol at any time during an interruption in the routine.

    II. CLEARING

      A. Clearing Agents
      There are many clearing agents in use. often the choice depends on the whim of the technician. it is desirable to use an agent that does not harden the tissue, that clears properly, and that is miscible with embedding paraffin as well as with ethanol. The clearing agent serves as a transition medium between two immiscible compounds, ethanol and paraffin.

      1. Xylene Commonly used, tends to harden tissue if left in too long, neurotoxic (hangover!)
      2. Benzene or toluene. Commonly used; clears overnight.
      3. Cedarwood oil. Slightly slower in penetrating than benzene is; does not cause hardening; does not interfere too seriously with paraffin penetration if it is not completely removed.
        1. 100% ethanol: cedarwood oil (1:1), 1 to 2 hours.
        2. Fresh cedarwood oil; overnight or longer to clear.
        3. Tissues can be left in cedarwood oil indefinitely. It does not harden the tissue.
      4. Methyl benzoate.Very good for clearing; does not harden tissues; avoids use of xylene; benzene is used to rinse out the clearing agent. It penetrates almost as fast as does cedarwood oil (12 to 24 hours) and is most valuable with the Peterfi celloidin-impregnation technique.
      5. Dioxane.Many directions and techniques make use of dioxane, which is miscible both with water and paraffin. It is used primarily when time is important because the tissues may be embedded with paraffin within 4 hours after fixation. The tissues are transferred to dioxane straight from Bouin's fluid or a formalin fixative. The dioxane is changed 3 times within 4 hours and the tissues are transferred directly to paraffin (3 changes are made in a total of 90 minutes). Dioxane causes greater shrinkage than xylene does. In addition, it is dangerous. Fumes of dioxane are toxic to humans; reportedly it is liver poison. Dioxane must be used in a hood at all times and must be stored in tightly sealed jars.

    III. INFILTRATION

      Never leave a tissue in the paraffin oven for more than 4 hours. The shorter the time in the hot oven with adequate paraffin impregnation and evaporation of clearing agent, the better for the tissue. Tissues become increasingly harder and more brittle as they are heated. Generally, use Paraplast with a melting point of 56 to 58 degrees Celsius.
        During the winter 54 to 56 degrees Celsius Paraplast may be used if the tissue is cut in a cool room.
        During the summer it may be necessary to use 60 to 63 degrees celsius Paraplast. This is to be avoided if possible in order to not to "cook" the tissue. "Cooked" tissue does not section or if it does, it does not stain well and most details are destroyed.
        Bioloid paraffin (a mixture of paraffin and other waxes) and Tissuemat are also used
        1. After Clearing Transfer to a mixture of xylene (benzene) saturated with Paraplast (paraffin and plastic polymers) chips. On top of
        2. the paraffin oven or in some warm spot at about 35 degrees Celsius, for 15 minutes.
        3. Infiltrate with hot paraplast in a 58 degree celsius paraffin oven in an open container.
        4. For 3 changes with the following time sequence: 30 minutes, 30 minutes, 1 hour.

    IV. EMBEDDING

      The tissue is now going to be placed in a mold and cooled. The choice of mold will depend on the type of chuck in the microtome you will use to section the tissue. Stainless steel, ceramic, paper, plastic, and aluminum foil molds can be used. The basic method is the same for each.
      1. Spray a stainless steel mold with releasing compound then pour a small amount melted Paraplast (fresh) into it. This will be demonstrated.
      2. Transfer the tissue with hot forceps and orient it properly in the center of the depression.
      3. Place the white plastic form on top of the mold and fill with melted paraplast. Be sure that the form is labeled. If a box or dish method is used insert a paper label to mark the point of orientation.
      4. Remove from heat.
      5. Cool the top surface of the Paraplast by blowing gently on it. Tissues at this stage are very brittle; handle
      6. them with care. As soon as a scum of Paraplast has formed on top, sink the cup gently into a cold water bath or place it on a refrigerated surface.
      7. The block will be ruined if it is submerged before its upper surface has formed a protective scum. Cool thoroughly in cold running
      8. tap water. If you use ice water for the final cooling, you may split the block owing to too rapid shrinkage. Paraplast naturally splits in the line of least resistance-right through the tissue. If you use plastic cups, the Paraplast block can be removed as soon as it is cooled. The stainless steel mold should slip off easily when cool and can be used again.
      Orientation of tissues in the Paraplast block is important for tissues such as duodenum when sections in a predetermined plane, such as cross sections, are required. Also, trimming is excessively difficult in a block embedded with two or more tissues if they are not carefully lined up before the Paraplast is cooled.

    Rain_line

    Laboratory Handouts
    Use your Browser "Back" button to return to your Laboratory

    To Return to:
    Histology Lecture Topics