A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue
Imaging mass cytometry (IMC) is able to quantify the expression of dozens of markers at sub-cellular resolution on a single tissue section by combining a novel laser ablation system with mass cytometry. As such, it allows us to gain spatial information and antigen quantification in situ, and can be...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-07-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fimmu.2020.01466/full |
| _version_ | 1818440951480188928 |
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| author | Nannan Guo Vincent van Unen Vincent van Unen Marieke E. Ijsselsteijn Laura F. Ouboter Andrea E. van der Meulen Susana M. Chuva de Sousa Lopes Noel F. C. C. de Miranda Frits Koning Na Li Na Li |
| author_facet | Nannan Guo Vincent van Unen Vincent van Unen Marieke E. Ijsselsteijn Laura F. Ouboter Andrea E. van der Meulen Susana M. Chuva de Sousa Lopes Noel F. C. C. de Miranda Frits Koning Na Li Na Li |
| author_sort | Nannan Guo |
| collection | DOAJ |
| description | Imaging mass cytometry (IMC) is able to quantify the expression of dozens of markers at sub-cellular resolution on a single tissue section by combining a novel laser ablation system with mass cytometry. As such, it allows us to gain spatial information and antigen quantification in situ, and can be applied to both snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tissue sections. Herein, we have developed and optimized the immunodetection conditions for a 34-antibody panel for use on human snap-frozen tissue sections. For this, we tested the performance of 80 antibodies. Moreover, we compared tissue drying times, fixation procedures and antibody incubation conditions. We observed that variations in the drying times of tissue sections had little impact on the quality of the images. Fixation with methanol for 5 min at −20°C or 1% paraformaldehyde (PFA) for 5 min at room temperature followed by methanol for 5 min at −20°C were superior to fixation with acetone or PFA only. Finally, we observed that antibody incubation overnight at 4°C yielded more consistent results as compared to staining at room temperature for 5 h. Finally, we used the optimized method for staining of human fetal and adult intestinal tissue samples. We present the tissue architecture and spatial distribution of the stromal cells and immune cells in these samples visualizing blood vessels, the epithelium and lamina propria based on the expression of α-smooth muscle actin (α-SMA), E-Cadherin and Vimentin, while simultaneously revealing the colocalization of T cells, innate lymphoid cells (ILCs), and various myeloid cell subsets in the lamina propria of the human fetal intestine. We expect that this work can aid the scientific community who wish to improve IMC data quality. |
| first_indexed | 2024-12-14T18:20:31Z |
| format | Article |
| id | doaj.art-51e04bb2c3e747548d589002420b860c |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-12-14T18:20:31Z |
| publishDate | 2020-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-51e04bb2c3e747548d589002420b860c2022-12-21T22:52:05ZengFrontiers Media S.A.Frontiers in Immunology1664-32242020-07-011110.3389/fimmu.2020.01466548984A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen TissueNannan Guo0Vincent van Unen1Vincent van Unen2Marieke E. Ijsselsteijn3Laura F. Ouboter4Andrea E. van der Meulen5Susana M. Chuva de Sousa Lopes6Noel F. C. C. de Miranda7Frits Koning8Na Li9Na Li10Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, NetherlandsImmunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, NetherlandsInstitute for Immunity, Transplantation and Infection, Stanford University, Stanford, CA, United StatesPathology, Leiden University Medical Center, Leiden, NetherlandsGastroenterology, Leiden University Medical Center, Leiden, NetherlandsGastroenterology, Leiden University Medical Center, Leiden, NetherlandsAnatomy, Leiden University Medical Center, Leiden, NetherlandsPathology, Leiden University Medical Center, Leiden, NetherlandsImmunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, NetherlandsImmunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, NetherlandsKey Laboratory of Zoonoses Research, Ministry of Education, Institute of Zoonoses, College of Veterinary Medicine, Jilin University, Changchun, ChinaImaging mass cytometry (IMC) is able to quantify the expression of dozens of markers at sub-cellular resolution on a single tissue section by combining a novel laser ablation system with mass cytometry. As such, it allows us to gain spatial information and antigen quantification in situ, and can be applied to both snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tissue sections. Herein, we have developed and optimized the immunodetection conditions for a 34-antibody panel for use on human snap-frozen tissue sections. For this, we tested the performance of 80 antibodies. Moreover, we compared tissue drying times, fixation procedures and antibody incubation conditions. We observed that variations in the drying times of tissue sections had little impact on the quality of the images. Fixation with methanol for 5 min at −20°C or 1% paraformaldehyde (PFA) for 5 min at room temperature followed by methanol for 5 min at −20°C were superior to fixation with acetone or PFA only. Finally, we observed that antibody incubation overnight at 4°C yielded more consistent results as compared to staining at room temperature for 5 h. Finally, we used the optimized method for staining of human fetal and adult intestinal tissue samples. We present the tissue architecture and spatial distribution of the stromal cells and immune cells in these samples visualizing blood vessels, the epithelium and lamina propria based on the expression of α-smooth muscle actin (α-SMA), E-Cadherin and Vimentin, while simultaneously revealing the colocalization of T cells, innate lymphoid cells (ILCs), and various myeloid cell subsets in the lamina propria of the human fetal intestine. We expect that this work can aid the scientific community who wish to improve IMC data quality.https://www.frontiersin.org/article/10.3389/fimmu.2020.01466/fullimaging mass cytometryIMCsnap-frozen tissue sectionshuman intestinemass cytometry |
| spellingShingle | Nannan Guo Vincent van Unen Vincent van Unen Marieke E. Ijsselsteijn Laura F. Ouboter Andrea E. van der Meulen Susana M. Chuva de Sousa Lopes Noel F. C. C. de Miranda Frits Koning Na Li Na Li A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue Frontiers in Immunology imaging mass cytometry IMC snap-frozen tissue sections human intestine mass cytometry |
| title | A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue |
| title_full | A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue |
| title_fullStr | A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue |
| title_full_unstemmed | A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue |
| title_short | A 34-Marker Panel for Imaging Mass Cytometric Analysis of Human Snap-Frozen Tissue |
| title_sort | 34 marker panel for imaging mass cytometric analysis of human snap frozen tissue |
| topic | imaging mass cytometry IMC snap-frozen tissue sections human intestine mass cytometry |
| url | https://www.frontiersin.org/article/10.3389/fimmu.2020.01466/full |
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