An evaluation of enzymatic and heat epitope retrieval methods for the immunohistochemical staining of the intermediate filaments

Z. Fan, V. Clark, Raymond B Nagle

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7 Citations (Scopus)

Abstract

The ability to accurately demonstrate intermediate filament proteins (IFs) in formalin-fixed tissues is of great importance in the histodiagnosis of neoplasms owing to the highly conserved and cell lineage-specific pattern of expression of these proteins. In this study, we evaluated the use of enzymatic and heat epitope retrieval methods for improving the immunoreactivity of the IFs fixed at different time intervals in formalin. Normal tissue specimens were fixed in formalin for 4, 24, and 48 h and in 70% ethanol as controls. The IFs were reacted with the following monoclonal antibodies: anti-cytokeratin antibodies KA4 (reactive with cytokeratins 14, 15, 16, and 19) and 10.11 (reactive with cytokeratins 8 and 18), anti-vimentin, anti-desmin, anti-glial fibrillary acidic protein (GFAP, polyclonal), and anti-neurofilament (NF). All IFs had marked loss of reactivity with each monoclonal IF antibody after 24-48 h of fixation, except GFAP, which retained its reactivity with the polyclonal GFAP antibody used. The cytokeratin immunoreactivity for both anti-cytokeratin antibodies, KA4 and 10.11, was completely recovered by predigestion with protease 1 but was less effectively enhanced by microwave heating. The other IFs tested greatly benefited from heat epitope retrieval in citrate buffer (0.01 mol/L) at pH 6. Protease 1 worked less well for these IFs and completely destroyed vimentin and desmin reactivity. Unexpectedly, microwave heating of fixed specimens of kidney and liver in Tris buffer (0.5 mol/L) at pH 10 produced a strong, granular staining artifact that was due to the enhancement of avidin binding to endogenous biotin. This artifactual staining was successfully blocked by application of purified avidin before the addition of avidin-biotin-peroxidase complex. In conclusion, this study reemphasizes the importance of testing individual antibodies using the various conditions of fixation and epitope recovery.

Original languageEnglish (US)
Pages (from-to)49-58
Number of pages10
JournalApplied Immunohistochemistry
Volume5
Issue number1
DOIs
StatePublished - 1997

Fingerprint

Intermediate Filaments
Avidin
Keratins
Formaldehyde
Epitopes
Desmin
Hot Temperature
Vimentin
Staining and Labeling
Biotin
Microwaves
Heating
Anti-Idiotypic Antibodies
Keratin-15
Peptide Hydrolases
Monoclonal Antibodies
Keratin-14
Keratin-8
Keratin-18
Intermediate Filament Proteins

Keywords

  • biotin
  • epitope retrieval
  • immunohistochemistry
  • intermediate filaments

ASJC Scopus subject areas

  • Anatomy
  • Medical Laboratory Technology

Cite this

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title = "An evaluation of enzymatic and heat epitope retrieval methods for the immunohistochemical staining of the intermediate filaments",
abstract = "The ability to accurately demonstrate intermediate filament proteins (IFs) in formalin-fixed tissues is of great importance in the histodiagnosis of neoplasms owing to the highly conserved and cell lineage-specific pattern of expression of these proteins. In this study, we evaluated the use of enzymatic and heat epitope retrieval methods for improving the immunoreactivity of the IFs fixed at different time intervals in formalin. Normal tissue specimens were fixed in formalin for 4, 24, and 48 h and in 70{\%} ethanol as controls. The IFs were reacted with the following monoclonal antibodies: anti-cytokeratin antibodies KA4 (reactive with cytokeratins 14, 15, 16, and 19) and 10.11 (reactive with cytokeratins 8 and 18), anti-vimentin, anti-desmin, anti-glial fibrillary acidic protein (GFAP, polyclonal), and anti-neurofilament (NF). All IFs had marked loss of reactivity with each monoclonal IF antibody after 24-48 h of fixation, except GFAP, which retained its reactivity with the polyclonal GFAP antibody used. The cytokeratin immunoreactivity for both anti-cytokeratin antibodies, KA4 and 10.11, was completely recovered by predigestion with protease 1 but was less effectively enhanced by microwave heating. The other IFs tested greatly benefited from heat epitope retrieval in citrate buffer (0.01 mol/L) at pH 6. Protease 1 worked less well for these IFs and completely destroyed vimentin and desmin reactivity. Unexpectedly, microwave heating of fixed specimens of kidney and liver in Tris buffer (0.5 mol/L) at pH 10 produced a strong, granular staining artifact that was due to the enhancement of avidin binding to endogenous biotin. This artifactual staining was successfully blocked by application of purified avidin before the addition of avidin-biotin-peroxidase complex. In conclusion, this study reemphasizes the importance of testing individual antibodies using the various conditions of fixation and epitope recovery.",
keywords = "biotin, epitope retrieval, immunohistochemistry, intermediate filaments",
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AU - Fan, Z.

AU - Clark, V.

AU - Nagle, Raymond B

PY - 1997

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N2 - The ability to accurately demonstrate intermediate filament proteins (IFs) in formalin-fixed tissues is of great importance in the histodiagnosis of neoplasms owing to the highly conserved and cell lineage-specific pattern of expression of these proteins. In this study, we evaluated the use of enzymatic and heat epitope retrieval methods for improving the immunoreactivity of the IFs fixed at different time intervals in formalin. Normal tissue specimens were fixed in formalin for 4, 24, and 48 h and in 70% ethanol as controls. The IFs were reacted with the following monoclonal antibodies: anti-cytokeratin antibodies KA4 (reactive with cytokeratins 14, 15, 16, and 19) and 10.11 (reactive with cytokeratins 8 and 18), anti-vimentin, anti-desmin, anti-glial fibrillary acidic protein (GFAP, polyclonal), and anti-neurofilament (NF). All IFs had marked loss of reactivity with each monoclonal IF antibody after 24-48 h of fixation, except GFAP, which retained its reactivity with the polyclonal GFAP antibody used. The cytokeratin immunoreactivity for both anti-cytokeratin antibodies, KA4 and 10.11, was completely recovered by predigestion with protease 1 but was less effectively enhanced by microwave heating. The other IFs tested greatly benefited from heat epitope retrieval in citrate buffer (0.01 mol/L) at pH 6. Protease 1 worked less well for these IFs and completely destroyed vimentin and desmin reactivity. Unexpectedly, microwave heating of fixed specimens of kidney and liver in Tris buffer (0.5 mol/L) at pH 10 produced a strong, granular staining artifact that was due to the enhancement of avidin binding to endogenous biotin. This artifactual staining was successfully blocked by application of purified avidin before the addition of avidin-biotin-peroxidase complex. In conclusion, this study reemphasizes the importance of testing individual antibodies using the various conditions of fixation and epitope recovery.

AB - The ability to accurately demonstrate intermediate filament proteins (IFs) in formalin-fixed tissues is of great importance in the histodiagnosis of neoplasms owing to the highly conserved and cell lineage-specific pattern of expression of these proteins. In this study, we evaluated the use of enzymatic and heat epitope retrieval methods for improving the immunoreactivity of the IFs fixed at different time intervals in formalin. Normal tissue specimens were fixed in formalin for 4, 24, and 48 h and in 70% ethanol as controls. The IFs were reacted with the following monoclonal antibodies: anti-cytokeratin antibodies KA4 (reactive with cytokeratins 14, 15, 16, and 19) and 10.11 (reactive with cytokeratins 8 and 18), anti-vimentin, anti-desmin, anti-glial fibrillary acidic protein (GFAP, polyclonal), and anti-neurofilament (NF). All IFs had marked loss of reactivity with each monoclonal IF antibody after 24-48 h of fixation, except GFAP, which retained its reactivity with the polyclonal GFAP antibody used. The cytokeratin immunoreactivity for both anti-cytokeratin antibodies, KA4 and 10.11, was completely recovered by predigestion with protease 1 but was less effectively enhanced by microwave heating. The other IFs tested greatly benefited from heat epitope retrieval in citrate buffer (0.01 mol/L) at pH 6. Protease 1 worked less well for these IFs and completely destroyed vimentin and desmin reactivity. Unexpectedly, microwave heating of fixed specimens of kidney and liver in Tris buffer (0.5 mol/L) at pH 10 produced a strong, granular staining artifact that was due to the enhancement of avidin binding to endogenous biotin. This artifactual staining was successfully blocked by application of purified avidin before the addition of avidin-biotin-peroxidase complex. In conclusion, this study reemphasizes the importance of testing individual antibodies using the various conditions of fixation and epitope recovery.

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