Abstract
BACKGROUND: Quantification of RNA expression and protein production in fluorescent stainings provides critical information concerning neurodevelopment. A trustable independent quantification technique requires acquisition of reliable images prior to image processing. There is uncertainty in existing literature regarding the use of confocal microscopy compared to standard epifluorescence microscopy, especially in the context of RNA in situ hybridization protocols. NEW METHOD: The hindbrains of developing rat embryos from embryologic day 14 (E14) to E20 were sectioned and stained for expression of Hoxb1, Hoxb2, and Phox2b using both RNAScope and immunohistochemistry. Islet1 was used for identification of hindbrain motoneuron cell bodies. Slides were imaged using both confocal and epifluorescence microscopy. RESULTS: Expression patterns of both mRNA and protein were similar in both imaging modalities. Analyses of Hoxb1 and Hoxb2 mRNA expression were particularly concordant between-scopes, with similar p-values and posthoc differences between timepoints. Confocal imaging of Hoxb2 protein yielded a significant peak at E18, but this level of significance was not reached using epifluorescence microscopy. Although similar trends were observed, only Phox2b RNAScope results were statistically significant when analyzed with confocal microscopy. In contrast, Phox2b immunostaining analyses showed significant differences using both microscopes. COMPARISON WITH EXISTING METHODS: Researchers may save time and financial resources if epifluorescence microscopy provides comparable or equal results as confocal. CONCLUSIONS: Epifluorescence microscopy appears sufficient for quantification of RNAScope experiments with relatively low puncta per cell, while confocal microscopy gives clearer definition to immunohistochemical protein relationships and may be preferable especially in targets with low protein production.