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Understanding complex pathophysiological processes involves studying intercellular responses and phenotypes within the organ microenvironment, preserving the spatial tissue architecture. This chapter explores a practical and cost-effective method for combining techniques such as in situ immunostaining and transcriptomics analysis. These protocols are adaptable to various mRNA targets, antibodies, tissue types, and tissue fixation appealing to a wide scientific community. We demonstrate their application in studying the host response to infection with a Salmonella enterica strain producing a toxin that induces DNA breaks. Specifically, we assessed the: (i) innate immune response to DNA breaks; (ii) co-detection of Salmonella mRNA fljB with the DNA damage marker γH2AX; (iii) co-detection of mRNAs for the cell cycle arrest marker p16INK4A and the proinflammatory and anti-inflammatory cytokines, Il6 and Il10, respectively. Considering that DNA damage is one of the leading causes of oncogene- and stress-induced-senescence, these protocols can be suitable to assess the cytokine profile associated with cellular phenotype and types of DNA damage of senescent cells in situ.

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Combined In Situ Transcriptomic and Immunofluorescence to Assess the Effects on DNA Damage and Innate Immune Responses Induced by Bacterial Genotoxin in In Vivo Models

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  • 发表日期：

    2025年

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  • Maria Lopez-Chiloeches,
  • Teresa Frisan

摘要

Understanding complex pathophysiological processes involves studying intercellular responses and phenotypes within the organ microenvironment, preserving the spatial tissue architecture. This chapter explores a practical and cost-effective method for combining techniques such as in situ immunostaining and transcriptomics analysis. These protocols are adaptable to various mRNA targets, antibodies, tissue types, and tissue fixation appealing to a wide scientific community. We demonstrate their application in studying the host response to infection with a Salmonella enterica strain producing a toxin that induces DNA breaks. Specifically, we assessed the: (i) innate immune response to DNA breaks; (ii) co-detection of Salmonella mRNA fljB with the DNA damage marker γH2AX; (iii) co-detection of mRNAs for the cell cycle arrest marker p16INK4A and the proinflammatory and anti-inflammatory cytokines, Il6 and Il10, respectively. Considering that DNA damage is one of the leading causes of oncogene- and stress-induced-senescence, these protocols can be suitable to assess the cytokine profile associated with cellular phenotype and types of DNA damage of senescent cells in situ.