J and Pereira.A. (Make reference to Stage 38 in the Step-By-Step Process) Video displaying information on how lower Parafilm pieces could be laid inside each well formulated with a small level of incubation option. This is certainly very important to guidelines with antibody and tyramide solutions specifically, which might be limited reagents inside our dual staining process. mmc2.mp4 (2.5M) GUID:?59F523DD-3D2D-4641-B54D-4CFFA7281DE4 Data Availability StatementNo datasets were generated nor analyzed in this scholarly research. Overview This process combines fluorescent hybridization and immunostaining to identify concurrently, in histological sections from the same animal, subpopulations of neurons activated after two episodes of sensory stimulation. It allows the identification of groups of cells singly activated by either stimulus or co-activated by both stimuli. Our method results in nuclear staining for mRNA and c-Fos protein, allowing better spatial and temporal resolution than previously published protocols, although it requires quick brain fixation. For complete details on the use and execution of this protocol, please refer to Carvalho et?al. (2015, 2020). Graphical Abstract Open in a separate window Before You Begin This dual staining protocol was devised to detect activated neurons in mouse brain sections, allowing the distinction between cells activated after each one of two sequential episodes of sensory stimulation (Carvalho et?al., 2015, 2020). Originally, our protocol was created to investigate, in the same animal, neurons in olfactory brain areas activated after stimulation with different chemosignals, but it is suited for the analysis of brain activity toward other types of sensory stimuli as well. Our method shares the same principles as the catFISH procedure (Guzowski et?al., 1999, 2001; Lin?et?al., 2011) but we employ newly designed probes to simultaneously detect mRNA and c-Fos protein (Carvalho et?al., 2015). The gene was chosen because it is a widely validated immediate early gene used as an indirect marker of neuronal activation in the brain, including in studies that focused DMP 696 on olfactory brain areas (Carvalho DMP 696 et?al., 2015; Lin et?al., 2011; Papes et?al., 2010). In our protocol, c-Fos protein is expressed in cells activated during the first window of sensory stimulation, while mRNA is produced in cells activated during the second window of stimulation, allowing the identification of cells activated by one, the other, or both stimuli, with great temporal resolution (Figure?1 and Methods Video S1). Open in a separate window Figure?1 Example of Dual c-Fos Staining and Controls (A) Top, time windows containing DMP 696 exposure to sensory stimuli, separated by 60?min of rest period. Bottom, example of microscopy image (maximum intensity projection in a z series of 20 confocal images). Green staining represents c-Fos protein labeling by immunostaining and red fluorescence indicates nuclear foci after mRNA detection by hybridization. Adapted from Carvalho et?al. (2015), under the Creative Commons Attribution License (CC BY). Scale bar, 50?m. (B) Single stimulation controls, showing low mRNA staining when DMP 696 stimulus is applied only in the first window and absence of c-Fos protein detection when stimulus is applied only in the second window. Data are represented as mean?+ SEM. Adapted from Carvalho et?al. (2020). Methods DMP 696 Video S1. Schematic Representation of Dual c-Fos Staining Method and Results (Refer to Microscopy Imaging section) The first segment shows the stimulation protocol and the image of a cell where c-Fos protein is expressed in FN1 the nucleus (green), representing activation during the first stimulation period. The second segment shows the stimulation protocol and a z-series depicting a cell where mRNA.
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