Multiple Immunoenzyme Staining: Methods and Visualizations for the Observation With Spectral Imaging

Abstract

Several staining concepts and color combinations exist to perform successful double immunoenzyme staining on human tissue specimens. Most of these concepts are based on differences between both primary antibodies: animal species, mouse Ig isotype or IgG subclasses, conjugates, or concentrations. Traditionally, double immunoenzyme staining has used chromogens selected to provide maximum color contrast when observed with the unaided eye. Unfortunately, visually good color combinations always include at least one diffuse chromogen, because of the paucity of appropriate chromogen colors. This situation is drastically changed with the use of spectral imaging, where multicolor microscopy can be unmixed in individual images based on their spectral characteristics. Spectral unmixing can be performed even up to quadruple immunoenzyme staining. This work contains practical suggestions for immunoenzyme double staining procedures for some frequently encountered primary antibody combinations: rabbit–mouse, goat–mouse, mouse–mouse, and rabbit–rabbit. The suggested protocols are all suitable for a classical red-brown color combination plus blue nuclear counterstain that is composed of peroxidase activity (diaminobenzidine tetrahydrochloride), alkaline phosphatase activity (Liquid Permanent Red), and hematoxylin, respectively. Although the red and brown chromogens do not contrast very well visually, they both show a crisp localization and can be perfectly unmixed by spectral imaging.

Influence of oral administration of HMB to pregnant dams on calbindin expression in the dentate gyrus of the hippocampus during postnatal development in spiny mice offspring

“…The measurements suggest a growing trend of the protein reactivity in offspring from dams treated with HMB. Moreover, it was demonstrated that the content of different antigens in cells may correlate with the DAB immunostaining intensity (15,22,28). In this case, the increase of immunostaining may indicate a change in the CB protein activity and Ca 2+ homeostasis in granular cells.…”

Delivery of bevacizumab to atheromatous porcine carotid tissue using echogenic liposomes

“…Using a custom image analysis routine, bevacizumab penetration was quantified in each arterial tunic by quantifying positively stained area in a binary fashion. This approach was undertaken due to the broadband, nonlinear absorption characteristics of DAB, which preclude use of the Beer-Lambert Law for quantifying stain intensity (van der Loos, 2008). Thus, the conclusions of this study are drawn from changes in the total area of tissue experiencing delivery of bevacizumab, and not the total amount of bevacizumab.…”

“…It is important to note that here, while the percentage of DAB-positive area was quantified in this study, the intensity of DAB-staining (weak, diffuse vs. strong, particulate) was not assessed, and varied significantly across the experimental samples. Future attempts to assess the intensity of DAB-positive staining as an indicator of the magnitude of local bevacizumab deposition should exercise caution regarding the non-linear optical density of DAB (van der Loos, 2008). …”

Decreased haemodynamic response and decoupling of cortical gamma-band activity and tissue oxygen perfusion after striatal interleukin-1 injection

“…Since DAB staining is not stoichiometric [23] and so cannot be compared between animals, this method was preferred because the contralateral hemisphere served as a control for individual animals. Any effect of oedema on the level of IgG immunostaining was diminished by averaging over the area of the whole hemisphere in each section.…”