Protocols

Tissue Preparation Protocols

Tissue Preparation Guidelines for Frozen and Paraffin blocks

The integrity of the antigen, as well as good tissue morphology, are the keys for a successful immunohistochemical study, and proper tissue preparation techniques serve as the foundation. Frozen and paraffin sections are used commonly in the facility. Each type of section follows a specific tissue preparation protocol. Following are some guidelines for two of the more popular methods, frozen and paraffin sectioning.

Frozen Tissue Preparation:

• This is the most common section type stained by the Facility, mostly due to the good maintenance of antigen integrity and tissue morphology
• Frozen sections can either be fixed or unfixed. The freezing protocol remains the same.
• For freezing tissues, you will need O.C.T. embedding compound (SAKURA), plastic tissue molds (Fisher), 2-methylbutane (Fisher), dry ice, a forceps or other clamping device, and a metal or plastic 1L container.
• Label the mold with proper tissue identification and fill partly with O.C.T.
• If freezing unfixed tissue, remove tissue from animal, dab it on a towel to remove any fluid, and immediately place in tissue mold with O.C.T. and continue with protocol
• If fixing first, remove tissue from animal, fix it, and then put in O.C.T. after go through cryo-protection procedure.
• Choose the best orientation for the tissue to be frozen in, and let it settle to the bottom. Note that the bottom of the mold is where cutting will begin.
• Add more O.C.T. on top of tissue to cover it completely and fill the mold.
• Fill the plastic or metal container half way with 2-methylbutane.
• Add several small pieces of dry ice and wait a few moments for the temperature to drop (-40C)
• Grasp the edge of the mold with the **forceps and dip into 2-methylbutane. Note that you should NOT submerge yet. Dip only the most bottom part of the plastic mold.
• The O.C.T. will begin to turn white as it freezes
• When all of the O.C.T. is frozen, drop the mold into the cold 2-methylbutane to freeze thoroughly (~5min)
• Remove the mold from the freezing liquid, wrap the mold in foil, label it, and immediately store at -80C.

Paraffin Tissue Preparation:

• Since more and more antigen retrieval methods have become available, paraffin sectioning has become more common and successful in the facility
• For paraffin sectioning, you will need to fix the tissue.
• Choose fixatives upon tissue type and antigen.  10% neutral buffered formalin and 4% paraformaldehyde are the fixatives commonly used in clinical and research laboratories.
• Tissue can be fixed at room temperature for 8-48 hours or longer depending upon the size and the tissue type.  It is recommended to cut tissue to certain size prior to fixation and processing.
• Other supplies you will need, include Histology Cassettes, a small sealable container, and 70%Ethanol.
• After proper fixation, wash the tissue with 0.1M PBS several times.
• Label the cassettes properly with pencil only.
• Place the cassette in the container filled with 70% Ethanol.
• Since the cassettes have small slits in them, it is a good idea to wrap small tissues (eg Lymph Nodes) in nylon mesh to prevent them from falling out
• Submit the samples to a histology or pathology laboratory.
• The processing and embedding paraffin blocks requires specialized equipment and expertise and it is usually preformed by a histology or pathology laboratory.

 Tissue Fixation Protocol

There are multiple fixatives used in immunohistochemistry, and some tissues require special fixation protocols. The following is a protocol for one of the most common type of fixative in immunohistochemistry laboratory, 4% paraformaldehyde and then embed it. You, however, should research what fixative is best for your tissue type, as well as the antibody you plan to use. Please contact the facility, or search for references using a scientific search engine (eg. Medline).

Tissue Fixation

• Dissect the tissues as quick as possible post mortem to preserve the morphology as well as possible
• Prepare fixative fresh each time right before use
• If using 4% paraformaldehyde, use the following recipe
• Prepare a 1x solution of PBS, and immediately before use
•  Heat the 0.1M PBS to 60C degree
• Weigh out the appropriate amount of paraformaldehyde to make a 4% solution and adding it to the warm 1x PBS
• IN A FUME HOOD, Slowly add drops of 1N NaOH until solution clears.
• Filter the fixative through Wattman's Paper if perfusion procedure is required, and adjust pH to 7.35-7.45
• Allow Fixative to cool to room temperature
• Put tissue into the fixative immediately after you dissect it.
• Use enough volumes of fixative.
• Fixation times may be varied depending upon tissue type and the size of the tissue.
• After fixation, Freezing of tissue block or Paraffin embedment can be done.