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

Abstract

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.

Keywords: affinity; antibody; elution; multiple immunostaining; stripping.

Figure 1.

Elution from tissue sections of antibodies as single layers or counterstained with secondary antibodies by three elution buffers. Serial sections of human colonic mucosa (top two rows) or tonsil (bottom two rows) were stained with the primary antibody alone (1st Ab) or primary and secondary antibodies (1st+2nd Ab), eluted with the buffers listed across the top, and then counterstained with an appropriate secondary or tertiary anti-hapten antibody sequence. (A–H) CD45 (K_D 3.6×10^-11); (I–P) Cytokeratin 7 (CK7) (K_D 2.1×10^-10). Note that Glycine (Gly) pH2 buffer fails to elute a counterstained CD45 antibody (G) and 6 M Urea (6MU) fails to elute a counterstained CK7 antibody (N). The faint background staining is due to prolonged NBT-BCIP development to ensure optimal sensitivity. Scale = 100 µm.

Figure 2.

Effect of Fab monomers vs whole Igs on the elution of high affinity antibodies. Tonsil sections were stained with CD45 rabbit monoclonal antibodies, secondary reagents and control-treated or eluted with 6 M Urea (6MU) or Glycine (Gly) pH2. CD45 was then re-applied in the case of Fab staining, followed by counterstaining. The staining sequence for each experiment is described in the referenced square. The absence of staining in (A), (B) and (D) is expected when the CD45 antigen in the tissue is masked by an existing primary antibody-Fab complex. Bar = 100 µm.

Figure 3.

Effect of the exposure to a 2-Mercaptoethanol, SDS buffer (2-ME/SDS) on the detection of tissue antigens. Paired serial sections were routinely stained for H&E and Periodic Acid Shiff, subjected to antigen retrieval, and then immunostained for a specific antigen/epitope (indicated on the left). Before staining, one (right column) of the two sections was exposed to the 2-ME/SDS buffer for 2 hr and 30 min, which is equivalent to five 30 min cycles. The tissues shown are kidney (top two rows) and tonsil (the remaining rows). Bar = 100 µm.

Figure 4.

Elution of streptavidin bound to endogenous biotin by two elution buffers. 6 M Urea (6MU) elution (A) removes the granular endogenous staining in the kidney tubules, which remains evident (B) after 2-ME/SDS elution. Bar = 100 µm

Figure 5.

Routinely processed sections sequentially immunostained after elution and re-staining with diagnostic antibodies. One section of normal colon (left two images) and one section from a cutaneous nevus (right) are stained sequentially for the antigens named above each image. The primary antibodies have been counterstained with a multilink (mouse + rabbit) HRP-conjugated polymer. Note the clean background and no crossover staining left by the earlier staining. Bar = 100 µm.

Figure 6.

Multiple immunofluorescent staining of a human tonsil. The same tonsil section was stained three times, each with a three-combination set of primary antibodies, and counterstained with species-specific, fluorochrome-conjugated secondary antibodies. (A) Keratin (blue), Ki-67 (red) and IRF4 (green). (B) S100 (green), CD68 (red), DAPI (blue). (C) CD3 (red), CD20 (green), Ki-67 (blue). (D) CD68 (red), CD34 (green), Keratin (blue). The thumbnails on the right depict each of the nine stains obtained. Bar = 100 µm.