All animals used in this experiment were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN, USA). The animal procedures are approved by the Animal Care and Use Committee of East Tennessee State University, as well as complied with the NIH Guide for the Care and Use of Laboratory Animals. These animals were housed on a 12:12 h light cycle (lights on at 07:00 a.m.). Water and food were provided ad libitum. Rats were randomly assigned to different experimental groups after living an acclimation period of 5 days. The CSD paradigm is the same as reported previously (Chen et al. 2012). In brief, in a social defeat model, an adult male Fischer 344 rat (the “intruder”) was introduced in the home cage of a Long-Evans retired male breeder
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Brains were sectioned at 16 µm in a cryostat, mounted on Super-Frost Plus slides with embedding medium and stored at -80℃. Signals of ISH were detected by digoxigenin (DIG) labeled single strand cRNA probes. The DIG-labeled cRNA probes were transcribed in vitro from cDNAs of rat GR (~0.4 kb) and rat SERT (~0.7 kb) in pGEM-3Zf vectors with T7 RNA polymerase and DIG RNA labeling mix (Roche, Branford, CT, USA). The amount of the transcript was estimated by comparison to DIG-labeled control RNA (Roche, Branford, CT, USA) in Dot-blot assays on nylon membranes (Roche, Branford, CT, USA). The ISH protocol is similar to the one from Cold Spring Harbor Laboratory with minor modifications (Javelle et al. 2011). In brief, slides were fixed with 4% (w/v) paraformaldehyde followed by 0.05% (w/v) proteinase K treatment for 5 min at 21oC and acetylation with acetic anhydride. Lipids were extracted by washing through an ethanol series: 50, 70, 95 and 100% (vols). Pre-hybridized sections were incubated with 200 µl hybridization solution per slide containing 200 ng DIG-labeled probes at 55oC overnight. At the next morning, the sections were washed extensively and treated with RNase A, then proceed to block in 4% goat serum for one hour. The anti-DIG-alkaline phosphatase antibody (1:200 dilution, Roche, Branford, CT, USA) was applied to sections at 4oC overnight. The color was developed by NBT/BCIP substrates (Roche, Branford, CT, USA ) with refreshments twice a day for 3~5
Brains were sectioned at 16 µm in a cryostat, mounted on Super-Frost Plus slides with embedding medium and stored at -80℃. Signals of ISH were detected by digoxigenin (DIG) labeled single strand cRNA probes. The DIG-labeled cRNA probes were transcribed in vitro from cDNAs of rat GR (~0.4 kb) and rat SERT (~0.7 kb) in pGEM-3Zf vectors with T7 RNA polymerase and DIG RNA labeling mix (Roche, Branford, CT, USA). The amount of the transcript was estimated by comparison to DIG-labeled control RNA (Roche, Branford, CT, USA) in Dot-blot assays on nylon membranes (Roche, Branford, CT, USA). The ISH protocol is similar to the one from Cold Spring Harbor Laboratory with minor modifications (Javelle et al. 2011). In brief, slides were fixed with 4% (w/v) paraformaldehyde followed by 0.05% (w/v) proteinase K treatment for 5 min at 21oC and acetylation with acetic anhydride. Lipids were extracted by washing through an ethanol series: 50, 70, 95 and 100% (vols). Pre-hybridized sections were incubated with 200 µl hybridization solution per slide containing 200 ng DIG-labeled probes at 55oC overnight. At the next morning, the sections were washed extensively and treated with RNase A, then proceed to block in 4% goat serum for one hour. The anti-DIG-alkaline phosphatase antibody (1:200 dilution, Roche, Branford, CT, USA) was applied to sections at 4oC overnight. The color was developed by NBT/BCIP substrates (Roche, Branford, CT, USA ) with refreshments twice a day for 3~5