Tissue handling ■
Section handling ■
Epitope retrieval ■
Primary antibody ■ Visualization ■ Chromogene ■ Controls ■ Recommended control tissue
Most epitopes are masked (but not destroyed) in formalin fixed tissue and can be detected with antibodies after a proper pre-treatment of the sections. Heat induced epitope retrieval (HIER) has become the most important demasking method, applicable for more than 80% of all antibodies. The efficiency of HIER is largely a function of temperature, time, and pH and chemical composition of the buffer. Temperature and time are inversely related: 120°C in a pressure cooker for 5-10 min. roughly corresponds 100°C in a microwave oven for 20 min. or 60°C in an incubator for 24 hours. High temperature may cause damage to the morphology, especially in case of short fixation or prolonged heating.
Retrieval of many epitopes is also under the influence of pH. Four different main patterns of pH-influenced HIER is seen:
A. Good retrieval throughout the pH range (e.g., CD20, Ab clone L26),
B. Good retrieval at very low and at high pH but a profound decrease in the range pH 3-6 (e.g., Ki67, Ab clone MIB1, estrogen receptor, Ab clone 1D5),
C. Retrieval increasing with increasing pH (the large majority of epitopes),
D. Good retrieval at low pH only (e.g., Prion Protein, Ab 3F4, epithelial related antigen, Ab clone MOC-31).
With the exception of pattern D, a buffer at pH 8-9 is suitable for almost all epitopes. Still, however, HIER is carried out at pH 6 in most laboratories! In our experience, an increase in pH from 6 to 9 gives a four fold staining enhancement on the average. The best high pH buffer is Tris/EDTA (or EGTA) in combination. Addition of urea is advocated by some.
Tris/EDTA pH 9 (10/1 mM) buffer may be produced as follows:
1,21 g Tris, Merck 1.08387
0,37 g EDTA, Fluka Chemika 03680
1000 ml destilled water
Buffer pH is stable at 9.0 - 9.1, usually without adjustment. The buffer is stable for approximately 4 weeks at room temperature.
Special buffers like the DakoCytomation Target Retrieval Solution (TRS), pH 6, provide an advantage compared to the above mentioned alkaline buffers in a few selected cases (e.g., epithelial antigen, Ab clone Ber-EP4; CD31, Ab clone JC/70A; glycoprotein 200, Ab clone 66.4.C2; epithelial related antigen, Ab clone MOC-31).
The other major retrieval method is proteolysis. It has some drawbacks compared to HIER: The requirement for proteolysis is closely related to the fixation time (which is often not known). If the tissue has been shortly fixed, the pre-treatment should be diminished, otherwise the target proteins may be digested away. If on the other hand the fixation time has been prolonged, retrieval may be insufficient unless the proteolysis is enhanced. Antibodies for which the epitopes are better retrieved using proteolysis than HIER are, e.g., cytokeratin (CK) 8/7, Ab clone Cam 5.2; prostate specific antigen, Ab clone 28/A4; collagen IV, Ab clone CIV 22). Pronase is the enzyme most frequently used. Other enzymes like trypsin generally show no advantages. Pepsin seems particularly useful for extracellular epitopes (collagen IV, laminin).
Some epitopes are apparently demasked equally by HIER and proteolysis. Subtle differences may exist, however. Thus, using either of the methods S-100beta protein may be demasked with the same efficiency in nerves, whereas only HIER allow proper detection of S-100beta in some epithelia and striated muscle. Cytokeratin 20, Ab clone Ks20.8, give a false positive staining in some epithelia after proteolysis but not after HIER.
In some cases, where both heat and proteolysis provide a good epitope retrieval, the latter method may require a higher Ab concentration to give an optimal result.
Some laboratories has continued to use proteolytic pre-treatment, once accepted as a good method because of an apparently good control, even though HIER may be much more efficient. See for example a desmin staining based on the two different retrieval methods.
The need for pre-treatment is not always obvious. An example is the detection of CD117, using the polyclonal antibody from DakoCytomation (A4502), where we recommend HIER in an alkaline pH (see illustrations), while others state that the staining intensity is reduced after HIER (Hornick JL. Fletcher CDM Am J Clin Pathol 2002;117:188-193).