Chapter Fifteen - Preparation of Formalin-fixed Paraffin-embedded Tissue for Immunohistochemistry
Section snippets
Theory
Fixation: There are four general procedures for tissue fixation; snap freezing, OCT media embedding, RNA stabilizing reagent, or fixative. This protocol will focus on formalin fixation since it is the most common. There are multiple solutions that can be used for fixation of tissues, including Bouin's Fixative, paraformaldehyde, and neutral-buffered formalin. Tissue is fixed by cross-linkages between the lysine residues of proteins. The primary purpose of a fixative is to stop the progress of
Equipment
Tissue fixation cassettes
Beaker or container with lid (to submerge cassettes with enough room to fully cover them with liquid at a ratio of 10:1 fixative volume to tissue volume)
Automated Tissue Processor, if available
Paraffin warmer
Paraffin embedding mold
Water bath (35–40 °C)
Microtome
Oven (65 °C)
Razor blades
Forceps
Cutting board or surface (must be clean)
Materials
Formalin solution, neutral buffered, 10%
Sodium chloride (NaCl)
Potassium chloride (KCl)
Sodium phosphate dibasic (Na2HPO4)
Potassium phosphate monobasic (KH2PO4)
Ethanol (70%, 95%, and 100%)
Xylene
Paraffin
Preparation
Collect human tissue sample or laboratory animal tissues according to laboratory protocol and institutional guidelines.
Duration
Preparation Variable, depends on the time it will take for tissue collection Protocol 48 h (formalin fixation) ~ 24 h (processing) ~ 12 h (embedding) ~ 4–8 h (sectioning)
Embedding and sectioning times will vary greatly depending on the number of tissues and the skill of the scientist.
Caution
Work in a well-ventilated area and wear gloves when working with fixatives. Formalin contains formaldehyde,
Overview
The purpose of this step is to preserve the tissues with formalin so that they remain as biologically similar to when they were removed as possible.
Duration
48 h
- 1.1
As soon as you harvest a tissue, cut it to a thickness no greater than 3–4 mm using a sharp, clean razor blade and place specimen into a tissue fixation cassette.
- 1.2
Immediately put the cassette containing the tissue into a beaker containing formalin at a ratio of 10:1 fixative to tissue volume. Cover the beaker with two layers of foil. Alternatively
Overview
The purpose of this step is to dehydrate the tissue and embed it in a paraffin block. Most often this is done overnight using an automated tissue processor, but this protocol will use the same principles and can be performed without the machine.
Duration
3–4 h or overnight (for processing, using an automated tissue processor) a few hours (for paraffin to set up)
- 2.1
Place the tissues, while still in the cassettes, into 70% ethanol for 20 min.
- 2.2
Transfer the tissues into 95% ethanol for 20 min. Repeat incubation in
Overview
The purpose of this step is to make cuts or slices of the block, which contains the tissue embedded in paraffin, and mount these sections on glass microscope slides.
Duration
Will depend greatly on the skill of the user
- 3.1
Have a water bath ready at 35–40 °C.
- 3.2
Place the tissue block in a microtome, cryostat, or another sectioning device you have access to, so that it is parallel to the cutting blade.
- 3.3
Cut the tissues into slices between 4–5 μm in thickness.
- 3.4
Place the cut slices of tissue onto the surface of the
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