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Updated: Sep 1, 2025
Standardized Processing for Formalin-Fixed, Paraffin-Embedded Cell Pellet Immunohistochemistry Controls
Published on: July 27, 2022
Standardized Processing for Formalin-Fixed, Paraffin-Embedded Cell Pellet Immunohistochemistry Controls
Charles Havnar1, Kathy Hotzel1, Carmina Espiritu1
1Department of Pathology, Genentech.
Insights
Developing reliable immunohistochemistry (IHC) assays requires effective controls. This study presents a protocol for creating formalin-fixed, paraffin-embedded cell pellet controls for IHC method development, especially for novel proteins.
Area of Science:
- Biomedical Sciences
- Cell Biology
- Histotechnology
Background:
- Positive and negative controls are crucial for immunohistochemistry (IHC) assay development.
- Tissue controls are limited for novel or ubiquitously expressed proteins.
- Cell pellets offer standardized controls for antibody characterization.
Purpose of the Study:
- To describe a protocol for creating formalin-fixed, paraffin-embedded (FFPE) cell pellet controls.
- To provide a method for IHC assay development, particularly for novel proteins.
- To establish standardized controls for antibody selection and validation.
Main Methods:
- Processing and embedding cell pellets to mimic tissue processing.
- Utilizing cell lines with defined protein/transcript expression levels (high, medium, low).
- Incorporating engineered cell lines (over-expressing or gene-deleted via CRISPR).
Main Results:
- Successfully created FFPE cell pellet controls.
- Demonstrated the utility of cell pellets for IHC method development.
- Validated the recapitulation of tissue processing procedures.
Conclusions:
- FFPE cell pellet controls are valuable for IHC assay development, especially for challenging targets.
- This protocol facilitates the creation of standardized controls for antibody validation.
- Cellular controls enhance the reliability of IHC assays for research and diagnostics.
Abstract:
Positive and negative controls with known expression of target proteins are essential for the development of immunohistochemistry (IHC) assays. While tissue controls are beneficial for well-characterized proteins with defined tissue and cellular expression patterns, they are less suitable for the initial development of IHC assays for novel, poorly characterized, or ubiquitously expressed proteins. Alternatively, due to their standardized nature, cell pellets, including cancer cell lines with defined protein or transcript expression levels (e.g., high, medium, and low expression), transfected over-expressing cell lines, or cell lines with genes deleted through cell engineering technologies like CRISPR, can serve as valuable controls, especially for the initial antibody characterization and selection. In order for these cell pellets to be used in the development of IHC assays for formalin-fixed, paraffin-embedded tissues, they need to be processed and embedded in a manner that recapitulates the procedures used for tissue processing. This protocol describes a process for creating and processing formalin-fixed, paraffin-embedded cell pellet controls that can be used for IHC method developments.
