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 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.

Effects of antigen retrieval by microwave heating in formalin-fixed tissue sections on a broad panel of antibodies

1994 ◽  
Vol 102 (3) ◽  
pp. 165-172 ◽  
Author(s):  
R. von Wasielewski ◽  
M. Werner ◽  
M. Nolte ◽  
L. Wilkens ◽  
A. Georgii
Keyword(s):  
Microwave Heating ◽  
Antigen Retrieval ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Tissue Sections ◽  
Formalin Fixed

scholarly journals Detection of Streptococcus Suis by in Situ Hybridization, Indirect Immunofluorescence, and Peroxidase-Antiperoxidase Assays in Formalin-Fixed, Paraffin-Embedded Tissue Sections from Pigs

2000 ◽  
Vol 12 (3) ◽  
pp. 224-232 ◽  
Author(s):  
Mette Boye ◽  
Anne A. Feenstra ◽  
Conny Tegtmeier ◽  
Lars Ole Andresen ◽  
Søren R. Rasmussen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Indirect Immunofluorescence ◽  
Polyclonal Antibodies ◽  
Single Cells ◽  
Streptococcus Suis ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Streptococcus suis is an important pathogen in pigs and is considered a zoonotic agent. To aid diagnosis of infection caused by S. suis, a species-specific probe targeting 16S ribosomal RNA was designed and used for fluorescent in situ hybridization. Two additional immunohistochemical detection methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S. suis serotypes 1–31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from control tissue sections that contained organisms other than S. suis. These techniques are suitable for determining the in vivo localization of S. suis for research and diagnostic purposes.

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 Constant Detection of CD2, CD3, CD4, and CD5 in Fixed and Paraffin-embedded Tissue Using the Peroxidase-mediated Deposition of Biotin-Tyramide

1997 ◽  
Vol 45 (12) ◽  
pp. 1665-1672 ◽  
Author(s):  
Rainer Malisius ◽  
Hartmut Merz ◽  
Boris Heinz ◽  
Evariste Gafumbegete ◽  
Britta U. Koch ◽  
...  
Keyword(s):  
Binding Sites ◽  
Lymphoid Tissues ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Leukocyte Antigens ◽  
Formalin Fixed Paraffin Embedded ◽  
Tissue Specimens ◽  
Immunohistochemical Techniques ◽  
Microwave Techniques ◽  
Formalin Fixed

Immunohistochemical methods are widely used for diagnostic purposes in histopathology. However, the use of most monoclonal anti-leukocyte antibodies is limited to frozen tissues. Initially, it was believed that formalin fixation in particular, which is the gold standard for morphological tissue preservation, destroys most of the antigen binding sites. In recent years, protease digestion and the introduction of microwave techniques have significantly enhanced the sensitivity of immunohistochemical techniques, and a variety of hidden antigen sites in formalin-fixed tissue have been retrieved for initially unreactive antibodies. It therefore became clear that many of the leukocyte antigens are not irreversibly destroyed but are most probably masked during the fixation process. We developed a technique combining optimized pretreatment of formalin-fixed tissue with a dramatic enhancement of the immunohistochemical sensitivity and named it the ImmunoMax method. The ImmunoMax method proves that by optimizing the technique at the following three levels it is possible to detect formalin-sensitive leukocyte antigens: (a) standard fixation of the tissue; (b) sufficient antigen unmasking; and (c) increasing the substrate turnover by multiplication of binding sites with subsequent enhancement of the immunohistochemical reaction. Using this optimized ImmunoMax method, we were able to detect CD2, CD3, CD4, and CD5 with conventional monoclonal antibodies in formalin-fixed, paraffin-embedded tissue specimens of various lymphoid tissues.

scholarly journals Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue.

1988 ◽  
Vol 36 (5) ◽  
pp. 511-514 ◽  
Author(s):  
Y Hayashi ◽  
M Koike ◽  
M Matsutani ◽  
T Hoshino
Keyword(s):  
Human Brain ◽  
Enzymatic Digestion ◽  
Fixation Time ◽  
Reaction Products ◽  
Human Brain Tumor ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.

Standardization of Immunohistochemistry Based on Antigen Retrieval Technique for Routine Formalin-fixed Tissue Sections

1998 ◽  
Vol 6 (2) ◽  
pp. 89-96 ◽  
Author(s):  
Shan-Rong Shi ◽  
Richard J. Cote ◽  
Benjaporn Chaiwun ◽  
Lillian L. Young ◽  
Yan Shi ◽  
...  
Keyword(s):  
Antigen Retrieval ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Tissue Sections ◽  
Retrieval Technique ◽  
Formalin Fixed

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)

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