Pseudo-immunolabelling with the avidin-biotin-peroxidase complex (ABC) due to the presence of endogenous biotin in retinal Müller cells of goldfish and salamander.
Immunodetection techniques are dependent on enzyme-protein conjugates for the visualisation of antigen-antibody complexes. One of the most widely used is the avidin-biotin-peroxidase complex (ABC) method. The present study demonstrates that direct treatment of goldfish and salamander retinal sections with ABC, followed by an incubation with the chromogenic substrate 3,3-diaminobenzidine tetrahydrochloride (DAB) and H2O2, manifested a punctate staining pattern across the neural retinae, presumably through binding of avidin to endogenous biotin. Incubation with a primary antiserum against biotin followed by immunoprocessing with the peroxidase--anti-peroxidase (PAP) method showed a pattern similar to the punctuate framework as detected with solo ABC-treated sections. Moreover, the ABC-DAB/H2O2 mediated pattern corresponded to the spatial orientation of Müller cells as identified by GFAP immunostaining. These findings indicate the presence of endogenous biotin in Müller cells and calls for caution in the application of the ABC method in immunotechniques in retinal research.
Bhattacharjee
J
J
Graduate School Neurosciences, Amsterdam, The Netherlands.
Nunes Cardozo
B
B
Kamphuis
W
W
Kamermans
M
M
Vrensen
G F
GF
D000818:Animals; D001360:Avidin; D001710:Biotin / Q000032:analysis*; D015153:Blotting, Western; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis; D006054:Goldfish / Q000033:anatomy & histology / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D012160:Retina / Q000737:chemistry* / Q000166:cytology; D014562:Caudata / Q000033:anatomy & histology / Q000378:metabolism*
D005904:Glial Fibrillary Acidic Protein; D001360:Avidin; D001710:Biotin