Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon Cancer
Multiplex immunohistochemistry (mIHC) enables simultaneous staining of multiple immune markers on a single tissue section. Mounting studies have demonstrated the versatility of mIHC in evaluating immune infiltrates in different diseases and the tumour microenvironment (TME). However, the majority of...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-10-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/20/11001 |
| _version_ | 1797514369741881344 |
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| author | Lokman Pang Matthias Ernst Jennifer Huynh |
| author_facet | Lokman Pang Matthias Ernst Jennifer Huynh |
| author_sort | Lokman Pang |
| collection | DOAJ |
| description | Multiplex immunohistochemistry (mIHC) enables simultaneous staining of multiple immune markers on a single tissue section. Mounting studies have demonstrated the versatility of mIHC in evaluating immune infiltrates in different diseases and the tumour microenvironment (TME). However, the majority of published studies are limited to the analysis of human patient samples. Performing mIHC on formalin-fixed paraffin-embedded (FFPE) mouse tissues, particularly with sensitive antigens, remain challenging. The aim of our study was to develop a robust and reproducible protocol to uncover the immune landscape in mouse FFPE tissues. Effective antibody stripping while maintaining sensitivity to antigens and tissue adhesion to the glass slide is critical in developing an mIHC panel to allow successive rounds of staining. Thus, we identified a highly efficient stripping method that preserves signal intensity and antigenicity to allow multiple rounds of staining. We subsequently optimised an mIHC workflow with antibodies specific against CD4, CD8α, FOXP3 and B220 to identify distinct T and B cell populations on mouse FFPE tissues. Lastly, the application of this mIHC panel was validated in a mouse model of inflammatory bowel cancer, two allograft mouse models of spontaneous colon adenocarcinoma and a sporadic mouse model of colon cancer. Together, these demonstrate the utility of the aforementioned protocol in establishing the quantity and spatial localisation of immune cells in different pathological tissues. |
| first_indexed | 2024-03-10T06:30:39Z |
| format | Article |
| id | doaj.art-4908fff69a334051975ed073671cfc59 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T06:30:39Z |
| publishDate | 2021-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-4908fff69a334051975ed073671cfc592023-11-22T18:32:16ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-10-0122201100110.3390/ijms222011001Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon CancerLokman Pang0Matthias Ernst1Jennifer Huynh2Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, VIC 3084, AustraliaOlivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, VIC 3084, AustraliaOlivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Heidelberg, VIC 3084, AustraliaMultiplex immunohistochemistry (mIHC) enables simultaneous staining of multiple immune markers on a single tissue section. Mounting studies have demonstrated the versatility of mIHC in evaluating immune infiltrates in different diseases and the tumour microenvironment (TME). However, the majority of published studies are limited to the analysis of human patient samples. Performing mIHC on formalin-fixed paraffin-embedded (FFPE) mouse tissues, particularly with sensitive antigens, remain challenging. The aim of our study was to develop a robust and reproducible protocol to uncover the immune landscape in mouse FFPE tissues. Effective antibody stripping while maintaining sensitivity to antigens and tissue adhesion to the glass slide is critical in developing an mIHC panel to allow successive rounds of staining. Thus, we identified a highly efficient stripping method that preserves signal intensity and antigenicity to allow multiple rounds of staining. We subsequently optimised an mIHC workflow with antibodies specific against CD4, CD8α, FOXP3 and B220 to identify distinct T and B cell populations on mouse FFPE tissues. Lastly, the application of this mIHC panel was validated in a mouse model of inflammatory bowel cancer, two allograft mouse models of spontaneous colon adenocarcinoma and a sporadic mouse model of colon cancer. Together, these demonstrate the utility of the aforementioned protocol in establishing the quantity and spatial localisation of immune cells in different pathological tissues.https://www.mdpi.com/1422-0067/22/20/11001multiplex immunohistochemistrytumour microenvironmentimmune infiltrationimmune cellsinflammatory bowel diseasescolon cancer |
| spellingShingle | Lokman Pang Matthias Ernst Jennifer Huynh Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon Cancer International Journal of Molecular Sciences multiplex immunohistochemistry tumour microenvironment immune infiltration immune cells inflammatory bowel diseases colon cancer |
| title | Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon Cancer |
| title_full | Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon Cancer |
| title_fullStr | Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon Cancer |
| title_full_unstemmed | Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon Cancer |
| title_short | Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon Cancer |
| title_sort | development of a multiplex immunohistochemistry workflow to investigate the immune microenvironment in mouse models of inflammatory bowel disease and colon cancer |
| topic | multiplex immunohistochemistry tumour microenvironment immune infiltration immune cells inflammatory bowel diseases colon cancer |
| url | https://www.mdpi.com/1422-0067/22/20/11001 |
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