Tyramide signal amplification of immunofluorescent staining in mouse brain sections.

Mice were transcardially perfused with phosphate-buffered saline followed by 4% paraformaldehyde in phosphate buffer. Thirty µm serial sections were cut in a freezing microtome and transferred to phosphate-buffered saline. Free-floating sections were incubated with 1% hydrogen peroxide to quench endogenous peroxidase activity, blocked in 5% normal goat serum, then stained with a rabbit polyclonal antibody to calbindin D-28K (Chemicon) at a 1:1000 dilution. After washing, sections were incubated with Alexa Fluor® 488 goat anti–rabbit IgG antibody (Cat. No. A11008) at 5 µg/mL (upper panel) or HRP–goat anti–rabbit IgG antibody at 1 µg/mL, followed by Alexa Fluor® 488 tyramide (in TSA Kit #12, T20922; lower panel). Sections were washed, mounted on slides, coverslipped with ProLong® antifade reagent (in Kit P7481) and imaged under identical conditions (10× magnification, 250 millisecond exposure) using a bandpass filter set appropriate for fluorescein (FITC).

Mice were transcardially perfused with phosphate-buffered saline followed by 4% paraformaldehyde in phosphate buffer. Thirty µm serial sections were cut in a freezing microtome and transferred to phosphate-buffered saline. Free-floating sections were incubated with 1% hydrogen peroxide to quench endogenous peroxidase activity, blocked in 5% normal goat serum, then stained with a rabbit polyclonal antibody to calbindin D-28K (Chemicon) at a 1:1000 dilution. After washing, sections were incubated with Alexa Fluor® 488 goat anti–rabbit IgG antibody (Cat. No. A11008) at 5 µg/mL (upper panel) or HRP–goat anti–rabbit IgG antibody at 1 µg/mL, followed by Alexa Fluor® 488 tyramide (in TSA Kit #12, T20922; lower panel). Sections were washed, mounted on slides, coverslipped with ProLong® antifade reagent (in Kit P7481) and imaged under identical conditions (10× magnification, 250 millisecond exposure) using a bandpass filter set appropriate for fluorescein (FITC).

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