Antigen Retrieval Immunohistochemistry: Past, Present, and Future

Shi, Shan; Côté, Richard J.; Taylor, Clive R.

doi:10.1177/002215549704500301

Cited by 480 publications

(342 citation statements)

References 115 publications

(129 reference statements)

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“…For optimizing the staining results of all antibodies involved in double staining, four HIER solutions were tested (Shi et al 1997): citrate pH 6.0, EDTA pH 8.0, Tris-HCl 1 EDTA pH 9.0, and Tris-HCl pH 10.0 (Thermo Scientific/LabVision). After washing with running tap water, a non-serum protein block was applied for 15 min at room temperature (Ultra V Block; Thermo Scientific/LabVision).…”

Section: The Journal Of Histochemistry and Cytochemistrymentioning

confidence: 99%

Multiple Immunoenzyme Staining: Methods and Visualizations for the Observation With Spectral Imaging

Loos

2007

J Histochem Cytochem.

195

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Several staining concepts and color combinations exist to perform successful double immunoenzyme staining on human tissue specimens. Most of these concepts are based on differences between both primary antibodies: animal species, mouse Ig isotype or IgG subclasses, conjugates, or concentrations. Traditionally, double immunoenzyme staining has used chromogens selected to provide maximum color contrast when observed with the unaided eye. Unfortunately, visually good color combinations always include at least one diffuse chromogen, because of the paucity of appropriate chromogen colors. This situation is drastically changed with the use of spectral imaging, where multicolor microscopy can be unmixed in individual images based on their spectral characteristics. Spectral unmixing can be performed even up to quadruple immunoenzyme staining. This work contains practical suggestions for immunoenzyme double staining procedures for some frequently encountered primary antibody combinations: rabbit–mouse, goat–mouse, mouse–mouse, and rabbit–rabbit. The suggested protocols are all suitable for a classical red-brown color combination plus blue nuclear counterstain that is composed of peroxidase activity (diaminobenzidine tetrahydrochloride), alkaline phosphatase activity (Liquid Permanent Red), and hematoxylin, respectively. Although the red and brown chromogens do not contrast very well visually, they both show a crisp localization and can be perfectly unmixed by spectral imaging.

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Section: The Journal Of Histochemistry and Cytochemistrymentioning

confidence: 99%

Multiple Immunoenzyme Staining: Methods and Visualizations for the Observation With Spectral Imaging

Loos

2007

J Histochem Cytochem.

195

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“…Different heating methods have been used for antigen retrieval, such as autoclaving, pressure cooking, water bath, microwaving plus plastic pressure cooking, and steam heating. The temperature achieved by these methods appears to be the critical variable (47) .…”

Section: Limitations Of Predictive Factors Evaluation In Breast Cancementioning

confidence: 99%

“…However, antigen retrieval may cause false-positive stains or nonspecific background due to endogenous biotin. Although problems with endogenous biotin have previously been negligible in formalin-fixed tissue, antigen demasking also makes endogenous biotin more accessible (5,47) . High temperature, long heating time and high pH of the retrieval solution also increase the reactivity of endogenous biotin although they give the most efficient epitope retrieval (5,47) .…”

Section: Limitations Of Predictive Factors Evaluation In Breast Cancementioning

confidence: 99%

“…Although problems with endogenous biotin have previously been negligible in formalin-fixed tissue, antigen demasking also makes endogenous biotin more accessible (5,47) . High temperature, long heating time and high pH of the retrieval solution also increase the reactivity of endogenous biotin although they give the most efficient epitope retrieval (5,47) . In our experience, the antigen retrieval for ER and PR using high pH (EDTA, pH = 9) increases the reactivity, but also produces more non-specific and background staining.…”

Section: Limitations Of Predictive Factors Evaluation In Breast Cancementioning

confidence: 99%

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Predictive factors of breast cancer evaluated by immunohistochemistry

Gobbi¹,

Nunes²

2008

J. Bras. Patol. Med. Lab.

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Hormone receptor and Her2 protein overexpression evaluated by immunohistochemistry (IHC) is widely validated as a predictive factor in breast cancer. The quality of the IHC reaction is influenced by tissue fixation and processing. Over-and underfixation deeply affect IHC results. Antigen retrieval may improve IHC but it does not recover tissue from autolysis or overfixation. The choice of primary antibody for IHC as to its sensitivity and specificity in relation to therapeutic response represents an important stage. Apart from mouse monoclonal antibodies, new rabbit monoclonal antibodies are commercially available, such as clones anti-ER SP1 and B644, anti-PR SP2 and B645 and anti-Her2 SP3 and 4B5. They represent an alternative to hormone receptor and Her2 evaluation by IHC. New polymeric nonbiotinylated detection systems are also available and allow accurate and strong marking with no stromal and no non-specific cytoplasmic staining due to endogenous biotin. The most recommended cut off for estrogen and progesterone receptors (ER and PR) is more than 1% of positive cells with moderate or strong staining intensity (Allred's scoring system). New guidelines for Her2 evaluation by IHC show a cut off of more than 30% of positive cells with strong intensity (3+) that correlates better with gene amplification. The 2+ cases are now considered indeterminate and should be confirmed by fluorescence in situ hybridisation (FISH)

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“…Recognition of antigens in tissue fixed by cross-linking agents is often improved by target retrieval using heat. 21 Following established protocols, we tried boiling the sections while immersed in citrate buffer in a microwave oven. This procedure led to a nearly complete loss of eGFP fluorescence that can probably be ascribed to the applied heat (the T m of wild-type GFP is 70°C) but not to the mildly acidic buffer (pH 6.0) because eGFP is reported to retain 50% of its fluorescence at this pH.…”

Section: In Situ Labeling Of Egfp ϩ Cells and Host Cells With Fluorocmentioning

confidence: 99%

In Situ Characterization of Genetically Targeted (Green Fluorescent) Single Cells and Their Microenvironment in an Adoptive Host

Leithäuser

Trobonjača

Reimann

et al. 2001

The American Journal of Pathology

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Stable expression of transgene-encoded enhanced green fluorescence protein (eGFP) was used as a sensitive and specific marker to detect in situ donor cells engrafted into different tissues of adoptive hosts. eGFP ؉ lymphoid or myeloid cells (eg, CD4 ؉ T cells or bone marrow-derived dendritic cells) from eGFPtransgenic C57BL/6 donor mice were injected into congenic, immunodeficient RAG1 ؊/؊ C57/BL6 hosts. eGFP ؉ cells were detected in the adoptive host from 2 days to 4 weeks after transfer using an optimized method of fixed cryopreservation to process the tissue. This allowed the simple, sensitive, and specific detection of eGFP ؉ donor cells in histological sections of transplanted hosts. We further demonstrate that this technique can be combined with other estab- have low sensitivity (often caused by high background reactivity of the tissue of interest), and may have limited specificity because of cross-reactivities with host cells that are difficult to control. The availability of monoclonal antibodies (mAbs) suitable for immunohistology in the murine system is limited. Hence, simple and specific techniques to identify in situ engrafted cells and to simultaneously determine their differentiation, activation, and viability status would greatly facilitate many studies in vivo.Green fluorescent protein (GFP) is a bioluminescent protein from the jellyfish Aequorea victoria. 3,4 Mutant variants of this protein have been developed that show enhanced fluorescence. A commonly used variant with enhanced fluorescence is GFPmut1 (designated eGFP in this article). 5 eGFP has been used as a reporter gene in prokaryotic and eukaryotic cells in vitro. A transgenic C57BL/6 mouse line has been constructed that expresses eGFP under the chicken ␤-actin promoter control. 6 -10 These mice have been used to study hemopoietic and lymphoid development, 11-13 spermatogenesis, 14 and maternal cell transmission to offspring through placenta during pregnancy and by breast-feeding after birth. 15 These studies required the isolation of viable cells from the host and their subsequent flow cytometric identification and characterization. Because of the lack of optimized techniques to detect eGFP-expressing cells in situ, an analysis of cell migration, proliferation, and differentiation by immunohistological techniques was difficult to perform in this attractive model.We transferred eGFP ϩ cells of well-defined lymphoid or myeloid subsets, ie, splenic CD4 ϩ T cells or bone marrow-derived dendritic cells (DCs), from eGFP-transgenic (eGFP-tg) C57/BL6 (B6) donor mice into congenic, immunodeficient RAG1 Ϫ/Ϫ B6 hosts. We used the stable expression of eGFP as a sensitive and highly specific

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Antigen Retrieval Immunohistochemistry: Past, Present, and Future

Cited by 480 publications

References 115 publications

Multiple Immunoenzyme Staining: Methods and Visualizations for the Observation With Spectral Imaging

Multiple Immunoenzyme Staining: Methods and Visualizations for the Observation With Spectral Imaging

Predictive factors of breast cancer evaluated by immunohistochemistry

In Situ Characterization of Genetically Targeted (Green Fluorescent) Single Cells and Their Microenvironment in an Adoptive Host

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