Goat anti mouse igg coupled to alexafluor 488

Whole brains of rTg4510 and wild-type mice were fixed in cold 4% paraformaldehyde overnight, after which they were washed three times for 10 min with PBS solution. Sections (50 µm) were then prepared using a vibratome (Leica). Brain slices were incubated for 1 h in a blocking solution containing 0.5% BSA, 0.1% Triton X-100, and 0.05% sodium azide in PBS at RT. The slices were then incubated with mouse anti-NeuN antibody (1:500; Millipore) in blocking solution for 48 h at RT, after which they were washed again three times for 10 min with PBS and incubated in a secondary solution containing goat anti-mouse IgG coupled to AlexaFluor 488 (1:500; Life Technologies) in blocking solution for 3 h at RT. After a final three washes with PBS, the slices were cover-slipped with Vectashield mounting medium (H-1400; Vector Laboratories Inc.) and stored at 4 °C. To calculate CA1 neuronal densities the brain slices were imaged using a spinning-disk confocal system (Marianas; 3I, Inc.) consisting of an Axio Observer Z1 (Zeiss) equipped with a CSU-W1 spinning-disk head (Yokogawa Corporation of America) and an ORCA-Flash4.0 v2 sCMOS camera (Hamamatsu Photonics) with a 40× oil-immersion objective (1.4 NA; Zeiss).

Cells were seeded on coverslips 24 hr before treatment with SLs. At the indicated times, cells were treated with 0.7% Triton X-100 in Cytoskeletal buffer (10mM Hepes pH 7.0, 100mM NaCl, 300mM Sucrose, 3mM MgCl2, 0.7% Triton X-100), then fixed with 4% paraformaldehyde in PBS. The fixed cells were permeabilized using 0.1% Triton X-100 in PBS for 15 min followed by blocking with 10% goat serum and then incubation with primary antibodies. The bound antibodies were revealed by Goat-anti-mouse IgG coupled to Alexa Fluor 488 (Life Technologies) and goat anti-rabbit coupled to Alexa 594 (Life Technologies). After washes, the coverslips were mounted to slides using Vectashield (Vector Laboratories).