Simultaneous visualization and cell-specific confirmation of RNA and protein in the mouse retina

Andrew J. Stempel, Catherine Morgans, J. Timothy Stout, Binoy Appukuttan

    Research output: Contribution to journalArticle

    10 Citations (Scopus)

    Abstract

    Purpose: Simultaneous dual labeling to visualize specific RNA and protein content within the same formalin-fixed paraffn embedded (FFPE) section can be technically challenging and usually impossible, because of variables such as tissue fixation time and pretreatment methods to access the target RNA or protein. Within a specific experiment, ocular tissue sections can be a precious commodity. Thus, the ability to easily and consistently detect and localize cell-specific expression of RNA and protein within a single slide would be advantageous. In this study, we describe a simplified and reliable method for combined in situ hybridization (ISH) and immunohistochemistry (IHC) for detection of mRNA and protein, respectively, within the same FFPE ocular tissue.

    Methods: Whole mouse eyes were prepared for 5 micron FFPE sections after fixation for 3, 24, 48 or 72 h. Customized probes from Advanced Cell Diagnostics to detect mRNA for vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF-1α), and hypoxia-inducible factor 2-alpha (HIF-2α) were used for ISH. Various parameters were tested using the novel RNAscope method for ISH and optimized for compatibility with subsequent IHC for glial fbrillary acidic protein (GFAP) or GS-lectin within the same tissue section. Dual fuorescent visualization of Fast Red ISH and Alexa Fluor 488 IHC signal was observed with confocal microscopy.

    Results: A fixation time of 72 h was found to be optimal for ISH and subsequent IHC. The RNAscope probes for VEGF, HIF-1α, and HIF-2α mRNA all gave a strong Fast Red signal with both 48 h and 72 h fixed tissue, but the optimal IHC signal for either GFAP or GS-lectin within a retinal tissue section after ISH processing was observed with 72 h fixation. A pretreatment boiling time of 15 min and a dilution factor of 1:15 for the pretreatment protease solution were found to be optimal and necessary for successful ISH visualization with 72 h FFPE ocular tissue.

    Conclusions: The protocol presented here provides a simple and reliable method to simultaneously detect mRNA and protein within the same paraffn-embedded ocular tissue section. The procedure, after preparation of FFPE sections, can be performed over a 2-day or 4-day period. We provide an optimization strategy that may be adapted for any RNAscope probe set and antibody for determining retinal or ocular cell-specific patterns of expression.

    Original languageEnglish (US)
    Pages (from-to)1366-1373
    Number of pages8
    JournalMolecular Vision
    Volume20
    StatePublished - Sep 21 2014

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    In Situ Hybridization
    Retina
    RNA
    Formaldehyde
    Immunohistochemistry
    Proteins
    Hypoxia-Inducible Factor 1
    Messenger RNA
    Lectins
    Neuroglia
    Vascular Endothelial Growth Factor A
    Tissue Fixation
    Confocal Microscopy
    Peptide Hydrolases
    Antibodies

    ASJC Scopus subject areas

    • Ophthalmology
    • Medicine(all)

    Cite this

    Simultaneous visualization and cell-specific confirmation of RNA and protein in the mouse retina. / Stempel, Andrew J.; Morgans, Catherine; Timothy Stout, J.; Appukuttan, Binoy.

    In: Molecular Vision, Vol. 20, 21.09.2014, p. 1366-1373.

    Research output: Contribution to journalArticle

    Stempel, Andrew J. ; Morgans, Catherine ; Timothy Stout, J. ; Appukuttan, Binoy. / Simultaneous visualization and cell-specific confirmation of RNA and protein in the mouse retina. In: Molecular Vision. 2014 ; Vol. 20. pp. 1366-1373.
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    N2 - Purpose: Simultaneous dual labeling to visualize specific RNA and protein content within the same formalin-fixed paraffn embedded (FFPE) section can be technically challenging and usually impossible, because of variables such as tissue fixation time and pretreatment methods to access the target RNA or protein. Within a specific experiment, ocular tissue sections can be a precious commodity. Thus, the ability to easily and consistently detect and localize cell-specific expression of RNA and protein within a single slide would be advantageous. In this study, we describe a simplified and reliable method for combined in situ hybridization (ISH) and immunohistochemistry (IHC) for detection of mRNA and protein, respectively, within the same FFPE ocular tissue.Methods: Whole mouse eyes were prepared for 5 micron FFPE sections after fixation for 3, 24, 48 or 72 h. Customized probes from Advanced Cell Diagnostics to detect mRNA for vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF-1α), and hypoxia-inducible factor 2-alpha (HIF-2α) were used for ISH. Various parameters were tested using the novel RNAscope method for ISH and optimized for compatibility with subsequent IHC for glial fbrillary acidic protein (GFAP) or GS-lectin within the same tissue section. Dual fuorescent visualization of Fast Red ISH and Alexa Fluor 488 IHC signal was observed with confocal microscopy.Results: A fixation time of 72 h was found to be optimal for ISH and subsequent IHC. The RNAscope probes for VEGF, HIF-1α, and HIF-2α mRNA all gave a strong Fast Red signal with both 48 h and 72 h fixed tissue, but the optimal IHC signal for either GFAP or GS-lectin within a retinal tissue section after ISH processing was observed with 72 h fixation. A pretreatment boiling time of 15 min and a dilution factor of 1:15 for the pretreatment protease solution were found to be optimal and necessary for successful ISH visualization with 72 h FFPE ocular tissue.Conclusions: The protocol presented here provides a simple and reliable method to simultaneously detect mRNA and protein within the same paraffn-embedded ocular tissue section. The procedure, after preparation of FFPE sections, can be performed over a 2-day or 4-day period. We provide an optimization strategy that may be adapted for any RNAscope probe set and antibody for determining retinal or ocular cell-specific patterns of expression.

    AB - Purpose: Simultaneous dual labeling to visualize specific RNA and protein content within the same formalin-fixed paraffn embedded (FFPE) section can be technically challenging and usually impossible, because of variables such as tissue fixation time and pretreatment methods to access the target RNA or protein. Within a specific experiment, ocular tissue sections can be a precious commodity. Thus, the ability to easily and consistently detect and localize cell-specific expression of RNA and protein within a single slide would be advantageous. In this study, we describe a simplified and reliable method for combined in situ hybridization (ISH) and immunohistochemistry (IHC) for detection of mRNA and protein, respectively, within the same FFPE ocular tissue.Methods: Whole mouse eyes were prepared for 5 micron FFPE sections after fixation for 3, 24, 48 or 72 h. Customized probes from Advanced Cell Diagnostics to detect mRNA for vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF-1α), and hypoxia-inducible factor 2-alpha (HIF-2α) were used for ISH. Various parameters were tested using the novel RNAscope method for ISH and optimized for compatibility with subsequent IHC for glial fbrillary acidic protein (GFAP) or GS-lectin within the same tissue section. Dual fuorescent visualization of Fast Red ISH and Alexa Fluor 488 IHC signal was observed with confocal microscopy.Results: A fixation time of 72 h was found to be optimal for ISH and subsequent IHC. The RNAscope probes for VEGF, HIF-1α, and HIF-2α mRNA all gave a strong Fast Red signal with both 48 h and 72 h fixed tissue, but the optimal IHC signal for either GFAP or GS-lectin within a retinal tissue section after ISH processing was observed with 72 h fixation. A pretreatment boiling time of 15 min and a dilution factor of 1:15 for the pretreatment protease solution were found to be optimal and necessary for successful ISH visualization with 72 h FFPE ocular tissue.Conclusions: The protocol presented here provides a simple and reliable method to simultaneously detect mRNA and protein within the same paraffn-embedded ocular tissue section. The procedure, after preparation of FFPE sections, can be performed over a 2-day or 4-day period. We provide an optimization strategy that may be adapted for any RNAscope probe set and antibody for determining retinal or ocular cell-specific patterns of expression.

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