title: Simultaneous visualization and cell-specific confirmation of RNA and protein in the mouse retina.
abstract: OBJECTIVE
  Simultaneous dual labeling to visualize specific RNA and protein content within the same formalin-fixed paraffin 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 fibrillary acidic protein (GFAP) or GS-lectin within the same tissue section. Dual fluorescent 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 paraffin-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.
authors.affiliation: Eye and Vision Health, Flinders University, Adelaide, South Australia, Australia.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Stempel
authors.affiliation: Department of Physiology & Pharmacology, Oregon Health & Science University, Portland, OR.
authors.forename: Catherine W
authors.initials: CW
authors.lastname: Morgans
authors.affiliation: Cullen Eye Institute, Baylor College of Medicine, Houston, TX.
authors.forename: J Timothy
authors.initials: JT
authors.lastname: Stout
authors.affiliation: Eye and Vision Health, Flinders University, Adelaide, South Australia, Australia.
authors.forename: Binoy
authors.initials: B
authors.lastname: Appukuttan
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D005404:Fixatives; D005557:Formaldehyde; D015870:Gene Expression; D005904:Glial Fibrillary Acidic Protein; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008867:Microtomy; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D016612:Paraffin Embedding; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D016707:Tissue Fixation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D005136:Eye Proteins; D005404:Fixatives; D005557:Formaldehyde; D005904:Glial Fibrillary Acidic Protein; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009419:Nerve Tissue Proteins; D012313:RNA; D042461:Vascular Endothelial Growth Factor A; D000099162:Endothelial PAS Domain-Containing Protein 1; C497443:Hif1a protein, mouse; C528900:glial fibrillary astrocytic protein, mouse; C467480:vascular endothelial growth factor A, mouse