Alexa fluor 488 secondary antibody goat anti chicken igg

Tectal cryo-sections were washed three times in 1x PBS/0.1% Tween-20 (PBS-Tw) solution. For Anti-Reelin IHC antigen retrieval was performed: sections were pretreated with Sodium Citrate buffer (10 g sodium citrate, 30mL TX-100 into 1L PBS) for 5 min, heated at 75 C for 15 min and subsequently treated with a 1% SDS in PBS solution. Consequently, they were incubated 1 hr at room temperature in 10% Normal Goat Serum (Invitrogen) in PBS-Tw blocking solution followed by overnight incubation, at 4 C, with 1/200 dilution of mouse Reelin monoclonal antibody (Millipore). The Alexa Fluor 488 or Alexa Fluor 568 goat anti-mouse IgG (Life Technologies) and DAPI (Sigma) were diluted to a final concentration of 1/200 and 1/500 dilution respectively in blocking solution and were added for 2 hr at room temperature. Sections were washed five times in wash buffer (1x PBS/0.1% Tween-20) prior to addition of Vectashield drops for the coverslip placement. Slides were left at room temperature for 1 hr before microscopy analysis. For Anti-GFP IHC no antigen retrieval was performed and 1/500 dilution of chicken primary anti-GFP antibody (Genetex) and Alexa Fluor 488 secondary antibody goat anti-chicken IgG (1/500, Molecular probes) were used.