The secondary antibody solution is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Cross-adsorption or pre-adsorption is a purification step to increase specificity of the antibody resulting in higher sensitivity and less background staining. To minimize cross-reactivity, the goat anti-chicken IgY whole antibodies have been affinity-purified and cross-adsorbed against chicken serum containing non-immunoglobulin chicken serum proteins. Images were taken on an EVOS FL Auto 2 Imaging System (Product # AMAFD2000) with an Olympus 20X Super Apochromat objective (Product # AMEP4734) at 20X magnification. The image contains overlay of HuC/D (green) and beta-III-tubulin (red).
Cells were blocked with 3% BSA in PBS for 30 mins at RT, incubated with a HuC/D mouse monoclonal antibody (Product # A21271) at a dilution of 1:500 and a chicken anti beta-III-tubulin antibody at a dilution of 1:250 in 3% BSA in PBS for 1 hr at RT, washed 3X in PBS and then incubated with Invitrogen Alexa Fluor Plus 488 donkey anti-mouse IgG secondary antibody (Product # A32766) at a dilution of 1:1000 and Invitrogen Alexa Fluor Plus 555 goat anti-chicken IgY secondary antibody (Product # A32932) prepared in 3% BSA in PBS at a dilution of 1:500 for 1 hr at RT. Gibco Rat Cortex Neurons (Product # A1084001) were thawed and grown according to protocol using B-27 Plus Neurobasal Culture System (Product # A3653401) and GlutaMAX (Product # 35050061) for two weeks before processing with the Image-IT Fixation/Permeablization kit (Product # R37602) according to protocol. Immunofluorescent analysis of HuC/D and beta-III-Tubulin in Rat cortical neurons.