Multiple Immunostainings with Different Epitope Retrievals-The FOLGAS Protocol

Anna von Schoenfeld; Peter Bronsert; Michael Poc; Andrew Fuller; Andrew Filby; Stefan Kraft; Konrad Kurowski; Kristin Sörensen; Julia Huber; Jens Pfeiffer; Michele Proietti; Verena Stehl; Martin Werner; Maximilian Seidl

doi:10.3390/ijms23010223

Multiple Immunostainings with Different Epitope Retrievals-The FOLGAS Protocol

Int J Mol Sci. 2021 Dec 25;23(1):223. doi: 10.3390/ijms23010223.

Authors

Anna von Schoenfeld^{1

2}, Peter Bronsert^{1

3

4}, Michael Poc^{1

5}, Andrew Fuller⁶, Andrew Filby⁶, Stefan Kraft⁷, Konrad Kurowski^{1

3

4}, Kristin Sörensen¹, Julia Huber^{1

3}, Jens Pfeiffer^{8

9}, Michele Proietti^{10

11}, Verena Stehl¹², Martin Werner^{1

3

4}, Maximilian Seidl^{1

12}

Affiliations

¹ Institute for Surgical Pathology, Medical Center-University of Freiburg and Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany.
² Clinic for Internal Medicine, Berlin Jewish Hospital, 13347 Berlin, Germany.
³ Tumorbank Comprehensive Cancer Center Freiburg, University of Freiburg, 79106 Freiburg, Germany.
⁴ Core Facility for Histopathology and Digital Pathology, Medical Center-University of Freiburg and Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany.
⁵ Klinik Gut St. Moritz, 7500 St. Moritz, Switzerland.
⁶ Flow Cytometry Core Facility, Innovation, Methodology and Application Research Theme, Leech Building, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE1 7RU, UK.
⁷ Center for Dermatopathology, 79106 Freiburg, Germany.
⁸ Department of Oto-Rhino-Laryngology, Medical Center-University of Freiburg and Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany.
⁹ Praxis HNO am Theater, 79098 Freiburg, Germany.
¹⁰ Center for Chronic Immunodeficiency, Institute for Immunodeficiency, Medical Center-University of Freiburg and Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany.
¹¹ Klinik für Rheumatologie und Immunologie, Hannover Medical School (MHH), 30625 Hannover, Germany.
¹² Institute of Pathology, Heinrich Heine University and University Hospital of Duesseldorf, 40225 Duesseldorf, Germany.

Abstract

We describe a sequential multistaining protocol for immunohistochemistry, immunofluorescence and CyTOF imaging for formalin-fixed, paraffin-embedded specimens (FFPE) in the formalin gas-phase (FOLGAS), enabling sequential multistaining, independent from the primary and secondary antibodies and retrieval. Histomorphologic details are preserved, and crossreactivity and loss of signal intensity are not detectable. Combined with a DAB-based hydrophobic masking of metal-labeled primary antibodies, FOLGAS allows the extended use of CyTOF imaging in FFPE sections.

Keywords: CyTOF; crossreactivity; formalin; hydrophobic masking; immunofluorescence; immunohistochemistry; multiple staining; re-fixation; retrieval.

MeSH terms

Epitopes / chemistry*
Fixatives / chemistry
Fluorescent Antibody Technique / methods*
Formaldehyde / chemistry*
Humans
Immunohistochemistry / methods
Paraffin Embedding / methods*
Staining and Labeling / methods*
Tissue Fixation / methods

Substances

Epitopes
Fixatives
Formaldehyde

Abstract

MeSH terms

Substances

Grants and funding