Biotinylated anti rabbit igg

Paraffin sections were used for immunohistochemistry. Serial coronal sections of brain were gained and hippocampal sections were gathered in sequence. Every 15th staining section was chosen for quantitative analysis (five sections per rat). After being dewaxed in xylene, hippocampal sections were hydrated in a graded series of alcohols. Then the sections placed into 0.01 M citrate buffer (pH6.0) were heated for 8 min in microwave oven to complete antigen retrieval, subsequently were blocked for endogenous peroxidases. The treated hippocampal sections were sealed using 3% BSA for 30 min at room temperature, and were then incubated with an anti-α7nAChR antibody (0.5 mg/ml, Abcam, Cat. ab110851, United States ) overnight at 4°C. After washing three times, the sections were incubated with a biotinylated anti-rabbit IgG (1:1,000, Abcam, Cat.150,077, United States ) for 1 h at 37°C and were then developed via DAB reagent. Subsequently, the sections were counterstained with hematoxylin, dehydrated, and mounted on gelatin-coated slides. Images of CA1 and CA3 regions were obtained with 90× magnification. Three fields in one section were chosen for calculating the numbers of positive neurons. Positively-labeled neurons were quantified using ImageJ software.

The assay was modified from a previously described method [6] (link). Microtiter plates (96-well) were coated with 5-MTP standards or samples in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) at 4°C overnight. The coating buffer was removed and the plate was washed five times with 200 µl TBST. After the plate had been blocked with 100 µl of 1% BSA/TBS and washed, 100 µl of a rabbit polyclonal anti-5-MTP antibody (Abcam, Cambridge, UK.) was added to each well and incubated for 1.5 h at room temperature or overnight at 4°C. The antibody is highly specific for 5-MTP with little cross-reactivity with related compounds such as 5-hydroxytryptophan, L-tryptophan and 5-methoxytryptamine [6] (link). The plate was washed and treated with biotinylated anti-rabbit IgG (Abcam, Cambridge, UK) followed by streptavidin-HRP (Upstate, Temecula, CA). Tetramethylbenzidine (TMB) (Upstate) substrate mixture was added and the optimal density (O.D.) was analyzed at 450 nm and 570 nm. For each experiment, a standard curve was generated using pure 5-MTP.