Arc immunohistochemistry

A separate cohort of mice underwent fear conditioning, EtOH or control i.p. injections, and were presented with the 3-kHz novel tone stimulus (fear generalization test experiment), using identical procedures to those described above (Control n = 6 and EtOH n = 7). For a comparison of baseline or context-dependent Arc expression, another group (n = 4) was added that underwent fear conditioning and received control injections, but on the test day, explored context B for the identical amount of time as the treatment groups (EtOH and Control groups), but was not presented the novel 3-kHz tone (No tone group). Exactly 90 minutes following the fear generalization test (presentation of the 3-kHz novel tone or no tone control), mice were injected (i.p.) with a ketamine/xylazine cocktail solution (100:10 mg/mL) and transcardially perfused with ice cold 1X PBS, followed by ice cold 4% paraformaldehyde in 1X PBS (7.4 pH). The time point for harvesting the brain following cued fear memory retrieval for Arc immunohistochemistry (IHC) was based on several previous reports^31,32. Brains were removed and stored in 4% PFA overnight then transferred to 1X PBS and stored at 4 °C until vibratome sectioning (no longer than 4 days).

Brains were sectioned (40 µm) coronally on a vibratome (VT1200, Leica Biosystems Inc., Buffalo Grove, IL USA). Every other section was collected in a well plate (no more than 10 sections/well) in 1X PBS (7.4 pH) for free-floating immunohistochemistry. Immunohistochemical staining was counterbalanced across experimental conditions. Sections were rinsed in 1X PBS, then blocked in a 1X PBS/1% bovine serum albumin (BSA)/0.2% Triton-X solution for 30 min to reduce non-specific binding. Sections were then incubated for 24 hours on an orbital shaker in Arc (C-7) mouse monoclonal antibody (1:100) (Cat# sc-17839, RRID: AB_626696, Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature. The next day, sections were rinsed in 1X PBS before a 1-hour incubation on an orbital shaker in anti-mouse biotinylated IgG (1:200) (Vector Laboratories, Burlingame, CA USA) at room temperature. Sections were rinsed again in 1X PBS then incubated in an ABC kit (Vectastain, Vector Laboratories, Burlingame, CA USA) for 1 hour on an orbital shaker at room temperature. Sections were rinsed again in 1X PBS, and then incubated in DAB peroxidase substrate (Vector Laboratories, Burlingame, CA USA) for exactly 2 min. Sections were then rinsed in 1X PBS and mounted onto gel-coated slides. Sections were dehydrated first in a graded series of EtOH concentrations, and then in xylenes, before cover-slipping with DPX.