Sab4502128

We used a previously well-characterized affinity isolated polyclonal primary antibody raised in rabbit against amino acids 156–205 of the PDE4D protein (SAB4502128; Millipore Sigma Aldrich, Burlington, MA, United States) that recognizes human and rodent PDE4D based on sequence homology. The antibody is highly specific and detects endogenous levels of total PDE4D protein at a band migrating at ∼91 kDa. The antibody is suited for a range of applications, including immunohistochemistry, immunoblotting and ELISA as per manufacturer’s recommendations. The specificity and selectivity of the PDE4D antibody has been previously characterized using immunohistochemistry in myocytes to identify a role of PDE4D-PRKAR1α in cardiac contractility (Bedada et al., 2016 ). The primary antibody was used at 1:200 dilution and was complexed with rabbit-specific goat secondary antibodies. Normal sera and IgG-free BSA were purchased from Jackson ImmunoResearch (West Grove, PA, United States). All chemicals and supplies for electron microscopy were purchased from Sigma Aldrich (St. Louis, MO, United States) and Electron Microscopy Sciences (Hatfield, PA, United States), respectively.

For PDE4D and pS214-tau immunolabeling, all male three young (3 month) and three aged (27 to 29-month-old) Sprague-Dawley rats were used. Animals were anesthetized with Nembutal (50 mg/mL, i.p.) and perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB; pH7.4). After perfusion, brains were removed from the skull and immersed in 4% PFA overnight at 4°C. Coronal 60 μm-thick sections were then cut on a Vibratome (Leica V1000) and collected in 0.1 ml PB. Sections of the medial PFC (mPFC) were transferred for 1 h to Tris–buffered saline (TBS) containing 5% bovine serum albumin, plus 0.05% Triton X-100 to block non-specific reactivity, and incubated in rabbit PDE4D antibody (SAB4502128; Millipore Sigma Aldrich) at 1:200 dilution, or rabbit pS214-tau antibody (ab4846; Abcam) at 1:100 dilution, in TBS for 48 h at 4°C. The tissue sections were incubated in goat anti-rabbit biotinylated antibody (Vector Laboratories) at 1:300 in TBS for 2 h, and developed using the Elite ABC kit (Vector Laboratories) and diaminobenzidine (DAB) as a chromogen. Omission of the primary antibody eliminated all labeling. Sections were mounted on microscope slides and examined and photographed under an Axiophot microscope equipped with Axiocam camera (Zeiss).