Goat anti mouse cy3 secondary antibody

Following electrophysiological recording, slice cultures were processed for immunofluorescence labeling of parvalbumin (mouse anti-parvalbumin antibody 1:1000; Millipore, goat anti-mouse Cy3 secondary antibody (1:100; Millipore)) and DCF + cells filled with 0,1% Biocytin during electrophysiology were visualized using avidin-conjugated Alexa Fluor488.

Organotypic hippocampal slice cultures were fixed with paraformaldehyde 4%/sucrose 4% in phosphate-buffered saline (PBS) 0.1 M overnight at 4°C. After fixation, slice cultures were washed with PBS and detached from the insert membranes. Slices were processed free-floating in wells and rinsed with PBS between steps. Slices were exposed overnight to a solution containing 10% normal goat serum (GS) and Triton X 0.5%. Slices were incubated for three nights with the respective primary antibodies (anti-Myelin Basic Protein, MBP, 1:1000, ThermoFisher; anti-parvalbumin, PV, 1:1000, Millipore) in 0.3% Triton X/5% GS/PBS. This was followed by an overnight incubation (0.1% Triton X/3% GS/PBS) with a goat anti-rabbit Alexa488 or goat anti-mouse Cy3 secondary antibody (1:100; Millipore). Slices were mounted on gold-coated slides and coverslipped with Fluoromount-G Mounting Medium (SouthernBiotech).

Slice cultures were fixed with 4% paraformaldehyde/4% sucrose in phosphate-buffered saline (PBS) 0.1 M overnight at 4 °C and stored in 30% sucrose/PBS. For immunolabelling, slices were carefully detached from the PTFE membranes and processed free-floating. Incubation with a mouse derived anti-parvalbumin antibody (1:1000; Millipore) in 0.1% Triton X-100/PBS for 3 nights was followed by incubation over 24 hours with a goat anti-mouse Cy3 secondary antibody (1:100; Millipore). Slices were mounted on gold-coated slides and coverslipped with Vectashield HardSet Mounting Medium.