Disaccharides Protect Antigens from Drying-Induced Damage in Routinely Processed Tissue Sections.

Drying of the tissue section, partial or total, during immunostaining negatively affects both the staining of tissue antigens and the ability to remove previously deposited antibody layers, particularly during sequential rounds of de-staining and re-staining for multiple antigens. The cause is a progressive loss of the protein-associated water up to the removal of the non-freezable water, a step which abolishes the immunoavailability of the epitope. In order to describe and prevent these adverse effects, we tested, among other substances, sugars, which are known to protect unicellular organisms from freezing and dehydration, and stabilize drugs and reagents in solid state form in medical devices. Disaccharides (lactose, sucrose) prevented the air drying-induced antigen masking and protected tissue-bound antigens and antibodies from air drying-induced damage. Complete removal of the bound antibody layers by chemical stripping was permitted if lactose was present during air drying. Lactose, sucrose and other disaccharides prevent air drying artifacts, allow homogeneous, consistent staining and the reuse of formalin-fixed, paraffin-embedded tissue sections for repeated immunostaining rounds by guaranteeing constant staining quality in suboptimal hydration conditions.

A 2-Step Laemmli and Antigen Retrieval Method Improves Immunodetection.

Detection by immunohistochemistry of antigens relies on reproducibly optimal preanalytical and analytical variables such as fixation conditions, antigen retrieval (AR), and the resolutive power of the detection system. There is a need to improve immunodetection on routinely fixed and embedded material, particularly for scarcely represented but relevant antigens. We devised a 2-step method and applied it to a panel of antigens of common use for diagnosis, prognosis, individualized therapy use, or research. The first step consists of a 10 minutes. Incubation at 95°C with a modified Laemmli extraction buffer. This was followed by a traditional AR method. Detection of the vast majority of antigens was improved over a simple AR with preservation of tissue integrity, as shown by quantitative image analysis. The mechanism underlying the improved detection may be controlled denaturation followed by heat-mediated retrieval, a method we dubbed "antigen relaxing" and which will improve routine detection of scarce antigens in formalin-fixed, paraffin-embedded material.

Epitope recognition in the human-pig comparison model on fixed and embedded material.

The conditions and the specificity by which an antibody binds to its target protein in routinely fixed and embedded tissues are unknown. Direct methods, such as staining in a knock-out animal or in vitro peptide scanning of the epitope, are costly and impractical. We aimed to elucidate antibody specificity and binding conditions using tissue staining and public genomic and immunological databases by comparing human and pig-the farmed mammal evolutionarily closest to humans besides apes. We used a database of 146 anti-human antibodies and found that antibodies tolerate partially conserved amino acid substitutions but not changes in target accessibility, as defined by epitope prediction algorithms. Some epitopes are sensitive to fixation and embedding in a species-specific fashion. We also find that half of the antibodies stain porcine tissue epitopes that have 60% to 100% similarity to human tissue at the amino acid sequence level. The reason why the remaining antibodies fail to stain the tissues remains elusive. Because of its similarity with the human, pig tissue offers a convenient tissue for quality control in immunohistochemistry, within and across laboratories, and an interesting model to investigate antibody specificity.

Elution of High-affinity (>10-9 KD) Antibodies from Tissue Sections: Clues to the Molecular Mechanism and Use in Sequential Immunostaining.

Inconsistent results obtained with published methods for the elution of antibodies from tissue sections prompted the assessment of both old and new methods in combination with monoclonal rabbit antibodies of known, increased affinity (above 1×10(-9) KD). We tested an acidic (pH 2) glycine buffer, a 6 M urea hot buffer and a 2-Mercaptoethanol, SDS buffer (2-ME/SDS). Some antibodies were not removed by the glycine pH 2 or 6 M urea hot buffers, indicating that antibodies survive much harsher conditions than previously believed. We found that the elution is dependent upon the antibody affinity and is reduced by species-specific crosslinking via a dimeric or Fab fragments of a secondary antibody. The high affinity bond of exogenous streptavidin with the endogenous biotin can be removed by 6 M urea but not by the other buffers. 2-ME/SDS buffer is superior to glycine pH 2 and 6 M urea hot elution buffers for all antibodies because of its irreversible effect on the structure of the antibodies. It also has a mild retrieving effect on some antigens present on routinely treated sections and no detrimental effect on the immunoreactivity of the tissue. Therefore, 2-ME/SDS buffer is the method of choice to perform multiple rounds of immunostaining on a single routine section.