scholarly journals Antibody Identification for Antigen Detection in Formalin-Fixed Paraffin-Embedded Tissue Using Phage Display and Naïve Libraries

Antibodies ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 4
Author(s):  
Célestine Mairaville ◽  
Pierre Martineau
Keyword(s):  
Phage Display ◽  
Formalin Fixation ◽  
Antigen Retrieval ◽  
Alternative Strategies ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Antibody Libraries ◽  
Formalin Fixed

Immunohistochemistry is a widely used technique for research and diagnostic purposes that relies on the recognition by antibodies of antigens expressed in tissues. However, tissue processing and particularly formalin fixation affect the conformation of these antigens through the formation of methylene bridges. Although antigen retrieval techniques can partially restore antigen immunoreactivity, it is difficult to identify antibodies that can recognize their target especially in formalin-fixed paraffin-embedded tissues. Most of the antibodies currently used in immunohistochemistry have been obtained by animal immunization; however, in vitro display techniques represent alternative strategies that have not been fully explored yet. This review provides an overview of phage display-based antibody selections using naïve antibody libraries on various supports (fixed cells, dissociated tissues, tissue fragments, and tissue sections) that have led to the identification of antibodies suitable for immunohistochemistry.

scholarly journals Selection Strategies for Tissue-Specific Antibody Identification Using Phage Display and Naïve Libraries

2020 ◽  
Author(s):  
Célestine Mairaville ◽  
Pierre Martineau
Keyword(s):  
Phage Display ◽  
Antigen Retrieval ◽  
Alternative Strategies ◽  
Tissue Sections ◽  
Selection Strategies ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Antibody Libraries ◽  
Formalin Fixed

Immunohistochemistry is a widely used technique for research and diagnostic purposes that relies on the recognition by antibodies of antigens expressed in tissues. However, tissue processing and particularly formalin fixation affect the conformation of these antigens through the formation of methylene bridges. Although antigen retrieval techniques can partially restore antigen immunoreactivity, it is difficult to identify antibodies that can recognize their target especially in formalin-fixed paraffin-embedded tissues. Most of the antibodies currently used in immunohistochemistry have been obtained by animal immunization; however, in vitro display techniques represent alternative strategies that have not been fully explored yet. This review provides an overview of phage display-based antibody selections using naïve antibody libraries on various supports (fixed cells, dissociated tissues, tissue fragments, and tissue sections) that have led to the identification of antibodies suitable for immunohistochemistry.

scholarly journals Protein-embedding Technique: A Potential Approach to Standardization of Immunohistochemistry for Formalin-fixed, Paraffin-embedded Tissue Sections

2005 ◽  
Vol 53 (9) ◽  
pp. 1167-1170 ◽  
Author(s):  
Shan-Rong Shi ◽  
Cheng Liu ◽  
Jeanette Perez ◽  
Clive R. Taylor
Keyword(s):  
Solid Phase ◽  
Antigen Retrieval ◽  
Polymer Microsphere ◽  
Thin Sections ◽  
Tissue Sections ◽  
Embedding Technique ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Two Phases ◽  
Formalin Fixed

A serial study was performed to develop a protein-embedding technique for standardization of immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded (FFPE) tissue sections. A protein carrier matrix must have two phases, a liquid phase to allow uniform mixing of a protein and a solid phase forming a ‘block’ that can be fixed and processed in the same manner as human tissue. This standardized protein block would serve as a source of thin sections for control of IHC and therefore must also withstand the boiling conditions of antigen retrieval (AR). After multiple experiments, a method was developed utilizing polymer microsphere (beads) as a support medium for protein. The method showed particular promise for quantitative IHC.

scholarly journals Immunohistochemical detection of thrombospondin-1 in formalin-fixed, paraffin-embedded tissue.

1996 ◽  
Vol 44 (7) ◽  
pp. 761-766 ◽  
Author(s):  
G D Grossfeld ◽  
S R Shi ◽  
D A Ginsberg ◽  
K A Rich ◽  
D G Skinner ◽  
...  
Keyword(s):  
Transitional Cell ◽  
Antigen Retrieval ◽  
Cultured Fibroblasts ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Thrombospondin 1 ◽  
Formalin Fixed Paraffin Embedded ◽  
Carcinoma Of The Bladder ◽  
Immunohistochemical Techniques ◽  
Formalin Fixed

Thrombospondin-1 (TSP) is a 450-KD glycoprotein that was initially discovered in the platelet alpha-granule. It now appears that TSP is intimately involved in the regulation of a variety of cellular functions and cell-to-cell interactions. Recently, it has been demonstrated that TSP functions as a p53-dependent inhibitor of angiogenesis in cultured fibroblasts from Li-Fraumeni patients and therefore may be an important factor involved with tumor invasion and metastasis. It has previously been demonstrated that TSP can be detected in frozen tissue sections by immunohistochemical methods. Our objective in this study was to determine the optimal antigen retrieval (AR) protocol for detection of TSP in formalin-fixed, paraffin-embedded tissue by using tissue sections from patients with invasive transitional cell carcinoma of the bladder. The optimal AR protocol was determined utilizing a variety of heating conditions and antigen retrieval buffers. Our results demonstrate that TSP can be reliably detected in paraffin-embedded tissue by immunohistochemical techniques that utilize AR with high-temperature microwave heating and a low-pH Tris-HCI buffer. The importance of this method is that it allows the reliable detection of TSP in archival tissue. This should facilitate further investigation into TSP's role in the regulation of cellular processes, including its influence on tumor angiogenesis and metastasis.

scholarly journals Antigen Retrieval Immunohistochemistry: Past, Present, and Future

1997 ◽  
Vol 45 (3) ◽  
pp. 327-343 ◽  
Author(s):  
Shan-Rong Shi ◽  
Richard J. Cote ◽  
Clive R. Taylor
Keyword(s):  
Antigen Retrieval ◽  
Optimal Test ◽  
Enzymatic Pretreatment ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Further Development ◽  
Temperature Heating ◽  
Formalin Fixed ◽  
Selection Of

The antigen retrieval (AR) technique, which is predominantly based on high-temperature heating of tissues, is used as a non-enzymatic pretreatment for immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections. It has been widely applied in pathology and analytical morphology. The existence of a growing body of literature on the AR technique raises a number of interesting issues for the further development of AR. These issues include the use of a “test battery” and the concept of “maximal retrieval” applied to the selection of optimal test protocols for the standardization of AR. (J Histochem Cytochem 45:327–343, 1997)

scholarly journals Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

1991 ◽  
Vol 39 (6) ◽  
pp. 741-748 ◽  
Author(s):  
S R Shi ◽  
M E Key ◽  
K L Kalra
Keyword(s):  
Polyclonal Antibodies ◽  
Antigen Retrieval ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Pre Treatment ◽  
Immunohistochemical Techniques ◽  
Formalin Fixed

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.

scholarly journals Immunohistochemistry

2015 ◽  
Vol 5 (10) ◽  
pp. I
Author(s):  
G Aryal
Keyword(s):  
Tissue Section ◽  
Antigen Retrieval ◽  
Secondary Antibody ◽  
Basic Principles ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Frozen Tissue ◽  
Formalin Fixed Paraffin Embedded ◽  
Antigen Antibody ◽  
Formalin Fixed

Immunohistochemistry (IHC) or immunocytochemistry is a method of localizing specific antigen in tissue or cells based on antigen antibody reaction. IHC is the way of validating morphological findings. It helps in tumor diagnosis and classification, identify prognostic and predictive markers. IHC has a long history that dates back more than 70 years, when Coons1 first developed immunofluroscence technique to detect corresponding antigen in frozen tissue section. At oxford, Taylor and Burns2 developed the first successful demonstration of antigens in routinely processed formalin fixed paraffin-embedded sections. Since the early 1990s, IHC has been applied in routine formalin fixed paraffin embedded tissue. 3-4 Validation of reagents, protocols, controls and staining results are vital steps of IHC. The basic principles and protocols for fresh-frozen tissue sections are the same as those for paraffin sections, except that the antigen retrieval and dewaxing procedures are not required for frozen tissue sections. Titrations may also differ and must be separately optimized. Basic principles: Antigen-antibody recognition is based on the three-dimensional (3D) structure of protein or some other antigen, which may be compromised by formalin-induced modification of protein conformation (“masking”) but is restored in part by Antigen retrieval. Anti-A antibody binds specifically to antigen A in the tissue section. Antigen B (B) is depicted as a second antigenic determinant that is part of the anti-A molecule; anti-B antibody, made in a second species, will bind to this determinant. Thus anti-B, the so-called secondary antibody, can be used to locate the site of binding of anti-A, the primary antibody, in a tissue section. Basic IHC procedure Antigen retrieval (AR)-Enzymatic digestion (proteinase or trypsin), heat treatment (Microwave, water bath or autoclave) Blocking of non-specific background staining Incubation with primary antibody in humidity chamber Add avidin-biotin-peroxidase complex, which binds to secondary antibody Add 3, 3’ diaminobenzidine (DAB) as a chromagen (color changing reagent), with hematoxylin (Mayer's) counterstaining Theranostic Application IHC is becoming increasingly important for the evaluation of predictive markers that can help select patients who may respond to particular targeted therapies. Some of these CD117 for GI stromal tumors, Herceptin (Genentech, South San Francisco, CA) for HER2-positive breast cancers, and rituximab for CD20-positive lymphomas.

Sign in / Sign up

Export Citation Format

Share Document