Anti goat igg hrp conjugated antibody

Cortical neurons were cultured in 96-well plates (60,000 cells/well, 100 μL/well of medium, DIV8) and treated with C21 (0, 0.1, 1, 10 μM/15 min) and surface levels of TRKB was determined as previously described [23 (link),43 (link)]. Briefly, after drug administration, the wells were washed three times with cold PBS and fixed with 4% PFA for 20 min at room temperature (RT) under agitation. The cells were washed again three times with PBS for 5 min at RT and blocked for 1 h at RT (5% nonfat dry milk, 5% Bovine Serum Albumin-BSA-in PBS). Primary antibody against the extracellular portion of TRKB (R&D; #AF1494; 1:500) was added and incubated overnight (ON) at 4 °C. Following wash with PBS, the cells were incubated with anti-goat IgG HRP-conjugated antibody (Invitrogen; #61-1620; 1:5000) for 1 h at RT. The cells were washed four times with PBS (10 min at RT for each wash) and then ECL (1:1) was added to detect the signal by the plate reader (Varioskan Flash, Thermo Scientific, Waltham, MA, USA). The signal from the samples, after blank subtraction, were normalized by the average of the control group (C21 = 0) and expressed as percentage from control.