Viewrna 2 plex tissue assay kit

Mice were anesthetized using isoflurane, rapidly decapitated, and brains were rapidly extracted. Immediately after removal, the brains were placed on a square of aluminum foil on dry ice to freeze. Brains were then placed in a − 80°C freezer for no more than 1 week before slicing. Slices of the BNST (12 μm) were made on a Leica CM3050S cryostat (Germany) and placed directly on coverslips. FISH was performed using the Affymetrix ViewRNA 2-Plex Tissue Assay Kit with custom probes for 5-HT_1A and Cre designed by Affymetrix (Santa Clara, CA). Slides were coverslipped with Southern Biotech DAPI Fluoromount-G (Birmingham, AL). 3 × 5 tiled z stack (15 optical sections comprising 14 μ m total) images of the entire 12 μ m slice were obtained on a Zeiss 780 confocal microscope for assessment of 5-HT_1A and Cre expression. All images were preprocessed with stitching and maximum intensity projection. An image of the BNST from 3 mice in each condition was hand counted for each study using the cell counter plugin in FIJI (ImageJ).

Immediately after removal, brains were placed on a square of aluminum foil on dry ice to freeze for 5 minutes before wrapping to prevent tissue damage. Brains were then placed in a −80 °C freezer for no more than 1 week before slicing. In all, 12-μm slices containing the CeA and ovBNST were obtained on a Leica CM3050S cryostat (Leica Biosystems, Wetzlar, Germany) and placed directly on coverslips. FISH was performed using the Affymetrix ViewRNA 2-Plex Tissue Assay Kit with custom probes for Pdyn, Nts, Ntsr1, and Ntsr2 designed by Affymetrix (Santa Clara, CA, USA). FISH was also done using the Advanced Cell Diagnostics (ACD) HybEZ(TM) II Hybridization System with custom probes for Nts and Pdyn designed by ACD (Newark, CA, USA). Slides were coverslipped with SouthernBiotech DAPI Fluoromount-G. (Birmingham, AL, USA). z-Stack (3 × 5 tiled; 8 optical sections comprising 10.57 μm in total) were obtained on a Zeiss 800 confocal microscope. All images were preprocessed with stitching and maximum intensity projection. Quantification of probe colocalization was hand counted using the cell counter plugin in FIJI (ImageJ, NIH, Bethesda, MD, USA). For all studies, cells were classified into three groups: probe 1+, probe 2+ or probe 1 + and 2+. Co-localization ratio was calculated by (probe 1+ and probe 2+/probe 1+ or probe 2+)*100 (McCullough et al., 2018 ).