Tissue handling ■
Section handling ■
Epitope retrieval ■
Primary antibody ■ Visualization ■ Chromogene ■ Controls ■ Recommended control tissue
Select and test your antibody
For diagnostic purposes, a panel of approved antibodies should be selected. Each new Ab in the laboratory should be tested according to the producers instructions as well as the laboratory’s own standards and evaluated with respect to the clinical usefulness.
The optimal Ab concentration (i.e. maximal specific staining and minimal non-specific staining) is inversely related to incubation time and temperature. Compared to the most used incubation time, 30 min., incubation for 2 and 16 hours gives a two fold and four fold enhancement, respectively, on the average. Long incubation time not only saves Ab but also tends to give less background staining. Yet, it is less useful for diagnostic pathology. An incubation temperature of 37°C may enhance the Ab reaction but sometimes give more background staining as well, and increases the risk of drying out the sections.
Use the right detergent
Addition of detergents to the Ab diluent increases the penetration of the Ab but also in some cases may cause extraction of the antigen. Tween 20 seems to preserve some epitopes better than Triton X100.
Mouse serum and ascites fluid may contain natural
antibodies reacting with determinants expressed on certain human
glycoproteins expressed in many epithelial cell types(Gooi et al., 1982). Variable amounts of these antibodies
may be present in the ascites fluid of mice obtained by intraperitoneal
injection of cloned hybridomas. The contaminant antibodies can cause
difficulties in the interpretation of immunostainings. A number of
commercial available mouse ascites antibodies have been shown to contain
antibodies to blood group A carbohydrate determinants (Finstad et al.,
1991). The Golgi-associating of this glycoprotein has lead to the term
MAG (Mouse Ascites Golgi) (Spicer et al., 1994). MAG reaction is primary
seen in ascites from female mice (Kliman et al., 1995) and absorption
with blood group A human erythrocytes can eliminate the reaction (Finstad
et al., 1991, and Spicer et al., 1994). The reaction pattern is
illustrated in assessments of
Gooi et al. Natural antibodies as contaminants of hybridoma products. Biochem Biophys Res Commun (1982) vol. 106 (2) pp. 539-45.
Finstad et al. Some monoclonal antibody reagents (C219 and JSB-1) to P-glycoprotein contain antibodies to blood group A carbohydrate determinants: a problem of quality control for immunohistochemical analysis. J Histochem Cytochem (1991) vol. 39 (12) pp. 1603-10.
Kliman et al. A mucin-like glycoprotein identified by MAG (mouse ascites Golgi) antibodies. Menstrual cycle-dependent localization in human endometrium. Am J Pathol (1995) vol. 146 (1) pp. 166-81.
Spicer et al. Some ascites monoclonal antibody preparations contain contaminants that bind to selected Golgi zones or mast cells. J Histochem Cytochem (1994) vol. 42 (2) pp. 213-21.