BRIEF REPORT |
Correspondence to: Seok Hyung Kim, DiNonA Inc., 65 Umyeon-dong, Seo-cho Gu, Seoul, South Korea 137-140. E-mail: platoshkim@freechal.com
![]() |
Summary |
---|
![]() ![]() ![]() ![]() |
---|
An immunohistochemical method using biotinyl tyramine was recently introduced to amplify weak staining signals. Despite its high sensitivity, however, tyramine-based immunostaining has been limited by its increased background staining. In this study, to develop an improved protocol of biotinyl tyramine-based immunohistochemistry minimizing the background staining, we determined which staining steps lead to the nonspecific reaction and the most appropriate blocking agents for background-provoking steps. Trypton casein peptone and distilled water with Tween-20 were shown to be most effective as a blocking agent and a rinsing solution, respectively. In conclusion, we developed an optimized protocol for biotinyl tyramine-based immunohistochemistry with minimal background staining. (J Histochem Cytochem 51:129132, 2003)
Key Words: immunohistochemistry, tyramine, background staining
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() |
---|
THE SENSITIVITY of immunohistochemistry (IHC) has been improved by development of antigen retrieval (AR) methods (
Tissue specimens were collected from surgically dissected specimens submitted to the Department of Pathology at Seoul National University Hospital. All archival materials were routinely fixed in 10% neutral buffered formalin and embedded in paraffin. Sections (5-µm) were prepared on silane-coated slides (Sigma; St Louis, MO).
The reagents used to prepare the blocking solutions were as follows: nonfat dried (ND) milk (skim milk) from Beckton Dickinson (Sparks, MD); casein sodium salt, bovine serum albumin (BSA), and goat globulin from Sigma; and trypton casein peptone (TCP) from Amresco (Solon, OH). The immunostaining kit and all of antibodies (Abs), including anti-CD20 (L26), anti-ER (6F11), anti-CD3 (PS1), anti-vimentin (V9), anti-desmin (DE-R-11), anti-GFAP (GF-01), and anti-CD99 (DN16), were obtained from DiNonA (Seoul, Korea).
Tissue sections on microslides were deparaffinized with xylene, hydrated in serially diluted alcohol, and then immersed in 3% H2O2 to remove the endogenous peroxidase (HRP) activity. AR was done by heating the slides in 10 mM sodium citrate buffer (pH 6.0) in a microwave oven. Then the sections were incubated with primary Abs for 60 min and, after three successive rinsings with washing buffer, further incubated with biotinylated goat anti-mouse Abs (DiNonA) for 20 min. After rinsing, the tissue sections were incubated with HRP-conjugated streptavidin (SA-HRP) (DiNonA) for 20 min at room temperature (RT). The slides were washed and the chromogen was developed for 5 min with liquid 3,3'-diaminobenzidine (DAB) (DiNonA). The slides were then counterstained with Meyer's hematoxylin, dehydrated, and mounted with Canada balsam for examination.
Biotinyl tyramine was prepared as previously described elsewhere (
To determine which step was responsible for the background staining, we added three steps (Steps IV, III, and II) one after another to the conventional immunostaining method with only SA-HRP incubation (Steps V and VI) without primary Ab incubation and compared their background levels. Whereas the sole application of Step V and the addition of Step IV produced no and minimal background in lymph nodes, respectively, the successive addition of Steps III and IV induced intense nonspecific reactivity. Moreover, the additional application of Step II as the first step also yielded highly intense background staining. This nonspecific reactivity in lymph nodes was notably marked on lymphocyte cytoplasm, high endothelial venules, and attached adipose tissue.
To develop an optimized blocking agent, we tested which reagents block the background staining induced by the secondary Ab incubation step (Step II) and the SA-HRP incubation step (Step III), among 3 mg/ml goat globulin, 4% BSA, 4% casein sodium salt, 2% ND milk, or 8% TCP. When the various kinds of blocking agents were applied at Step III by diluting SA-HRP in PBS containing them, the use of 8% TCP almost eliminated the background staining in lymph nodes with adipose tissue. Moreover, 2% ND milk was also effective to a lesser extent, but the other reagents were ineffective. For Step II, a blocking test was performed, and the use of TCP, BSA, and ND milk showed notable if not complete elimination of background staining, with no difference among these agents. However, goat globulin and 4% casein sodium salt were revealed to be less effective. Therefore, to avoid misinterpretation due to Step III-related background staining, 8% TCP was used as the Step III blocking agent.
Various kinds of solutions, such as PBS (pH 7.3), Tris buffer (pH 7.0), citrate buffer (pH 5.0), imidazole buffer (pH 7.0), borate buffer (pH 8.0), and distilled water (DW), were tested to determine which rinsing solution is the most effective for reduction of background staining. Tween-20 was added to all these solutions as a detergent. Unexpectedly, DW with 0.03% Tween-20 showed remarkable reduction of background staining, wheras PBS, citrate buffer, and imidazole buffer were minimally effective, and borate buffer and Tris buffer were mildly effective.
On the basis of these results, we can suggest a modified protocol for biotinyl tyramine-based immunostaining, employing TCP as Step II and III blocking agent and DW with Tween-20 for the rinsing solution. To confirm the effectiveness of this modified protocol, we performed immunostaining of human lymph nodes with anti-CD20 Ab. With the conventional immunostaining method, the lymph nodes were stained with moderate intensity at 1:200 dilution, but no staining was observed at 1:10,000 dilution (Fig 1A). Biotinyl tyramine-based immunostaining without modification showed that lymph nodes were stained with the same intensity at 1:10,000, but with significant background staining (Fig 1B). However, our modified protocol for biotinyl tyramine-based immunostaining produced not only the same degree of staining intensity as that of the unmodified tyramine-based signal amplification method but also the drastically decreased background staining (Fig 1C). Furthermore, applied to other tissues and other primary Abs, this improved protocol showed remarkably decreased background staining without impairing the high sensitivity (Table 1).
|
|
In this study, we determined the cause of background staining in biotinylated tyramine-based IHC and exploited a novel modified protocol of the standard staining technique. The biotinylated Ab incubation step and the SA-HRP incubation step were critical for nonspecific background staining. The primary cause of this background staining may be the binding of SA-HRP to macromolecules through nonspecific ionic or hydrophobic interactions. The binding of SA-HRP to endogenous biotin or biotin-like proteins may be the second source of nonspecificity, because lymphoid tissue is known to be free of endogenous biotin (
In conclusion, our results show that a modified protocol of biotinyl tyramine-based IHC can minimize nonspecific background staining without compromising high sensitivity.
![]() |
Acknowledgments |
---|
Supported by a grant (2002) for Technical Renovation Development Enterprise from the Korea Small and Medium Business Administration.
We wish to thank Hee Jin Kim for helpful comments during the preparation of the manuscript.
Received for publication August 5, 2002; accepted September 18, 2002.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() |
---|
Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ (1989) Catalyzed reporter deposition, a novel method of signal amplification. J Immunol Methods 125:279-285[Medline]
Duhamel RC, Johnson DA (1985) Use of nonfat dry milk to block nonspecific nuclear and membrane staining by avidin conjugates. J Histochem Cytochem 33:711-714[Abstract]
Freedman LJ, Maddox MT (2001) A comparison of anti-biotin and biotinylated anti-avidin double-bridge and biotinylated tyramide immunohistochemical amplification. J Neurosci Methods 112:43-49[Medline]
Kerstens MJ, Poddighe PJ, Hanselaar AGJM (1995) A novel in situ hybridization amplification method based on the deposition of biotinylated tyramine. J Histochem Cytochem 43:347-352
Kim SH, Jung KC, Shin YK, Lee KM, Park YS, Choi YL, Oh KI et al. (2002) The enhanced reactivity of endogenous biotin-like molecules by antigen retrieval procedures and signal amplification with tyramine. Histochem J 34:97-103[Medline]
Mengel M, Werner M, von Wasielewski R (1999) Concentration dependent and adverse effects in immunohistochemistry using the tyramine amplification technique. Histochem J 31:195-200[Medline]
Merz H, Malisius R, Mannweiler S, Zhou R, Hartmann W, Orscheschek K, Moubayed P et al. (1995) Immunomax, a maximized immunohistochemical method for the retrieval and enhancement of hidden antigens. Lab Invest 73:149-156[Medline]
Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741-748[Abstract]
Vogt RF, Jr, Philips DL, Omar Henderson L, Whitfield W, Spierto FW (1987) Quantitative difference among various proteins as blocking agents for ELISA microtiter plates. J Immunol Methods 101:43-45[Medline]