Biotinyl tyramide stock solution

The sections were incubated with rabbit polyclonal antibody against MEnk (AB5026 from Millipore; 1:50,000, 1:500,000, or 1:5,000,000) or without the anti-MEnk antibody for 18 h in PBS-BSA. After several rinses in PBS, the sections were incubated with the polymer staining reagent (Histofine Simple Stain Kit; Nichirei, Tokyo, Japan) for 30 min. After several rinses in PBS, they were processed for tyramide signal amplification (TSA) using the TSA Biotin System (Perkin Elmer, Boston, MA, USA). The sections were incubated in Biotinyl Tyramide (amplification reagent) Working Solution that was made by diluting Biotinyl Tyramide Stock Solution (Perkin Elmer) 1:50 using 1X Plus Amplification Diluent (Perkin Elmer, FP1135) for 30 min. After several rinses in PBS, the sections were incubated with the Vectastain Elite ABC reagent (Vector) for 30 min in PBS. After several rinses in PBS, the bound peroxidase was visualized by incubating the sections with a solution containing 0.05% DAB and 0.01% H₂O₂ in 0.05M Tris-HCl (pH 7.4) for 10 min. After several rinses in water, the immunostained sections were dehydrated and cover-slipped with Malinol (Muto Pure Chemicals). Overview protocol for the PBTA-DAB staining is shown in Figure 1 (left).

The sections were incubated with a rabbit polyclonal antibody against PSD-95 (1:5,000; Cell Signaling) or a goat polyclonal antibody against Calbindin-D28K (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 18 h in PBS containing 3% BSA. After several rinses in PBS, the sections were incubated with the polymer-staining reagent (Histofine Simple Stain Kit; Nichirei) for 30 min. After several rinses in PBS, they were processed for TSA using the TSA Biotin System (Perkin Elmer). Sections were then incubated in the biotinyl tyramide amplification reagent. A working solution was prepared by diluting the Biotinyl Tyramide Stock Solution (Perkin Elmer) 1:50 using 1× Plus Amplification Diluent (Perkin Elmer) for 30 min. After several rinses in PBS, the sections were incubated for 30 min with the avidin-biotin-peroxidase complex (ABC) reagent from a Vectastain Elite ABC kit (Vector). The bound peroxidase was visualized by incubating the sections with a solution containing 0.05% 3,3′-diaminobenzidine (DAB; Merck, Darmstadt, Germany) and 0.01% H₂O₂ in 0.05 M Tris-HCl (pH 7.4) for 10 min. The immunostained sections were dehydrated and cover-slipped with Malinol (Muto Pure Chemicals, Tokyo, Japan).