Vibration enhancement of slide-mounted immunofluorescence staining.
Immunofluorescence techniques allow the determination of protein and small molecule distribution within tissues and individual cells. There have been important, innovative modifications of these techniques since their introduction to the biosciences including the use of a mounting medium that prevents photo-bleaching, non-ionic detergents to permeabilize membranes, multiple immunofluorescence labeling and antigen recovery techniques for optimizing ligand-target interactions. While methods have been optimized for ligand-target accessibility in free-floating sections, little innovation has occurred to improve antibody access and epitope recognition in immunohistochemistry on slide-mounted sections or cell culture. During our studies of brain signaling pathways, we sought to improve the immunofluorescence signal to noise ratio in these specimens. We present here a minor modification of immunofluorescence procedures that significantly increases antibody access to epitopes within tissue and improves staining quality while significantly shortening incubation time. Antibody-epitope interactions are dependent on access and affinity. Our technique is based upon application of a vibration source during antibody incubation which increases epitope access, shortens incubation time and thereby minimizes background immunofluorescence. Data are presented on benefits evident with several antibodies raised against proteins and peptides localized in various subcellular compartments. Analysis of the quality of labeling was performed to show that signal intensity is enhanced and background intensity is often diminished when incubations are performed under gentle vibration. This, together with the significant saving of time, should make this procedure applicable to a wide range of neurobiological questions.
Jacobsen
K X
KX
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ont., Canada. kjaco031@uottawa.ca
Staines
W A
WA
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology*; D016477:Artifacts; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D000939:Epitopes / Q000276:immunology*; D005455:Fluorescent Antibody Technique / Q000295:instrumentation / Q000379:methods*; D008297:Male; D009419:Nerve Tissue Proteins / Q000032:analysis; D009448:Neurochemistry / Q000295:instrumentation / Q000379:methods; D009474:Neurons / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D009479:Neuropeptides / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods*; D013347:Subcellular Fractions / Q000276:immunology; D013997:Time Factors; D014732:Vibration
D000906:Antibodies; D000939:Epitopes; D009419:Nerve Tissue Proteins; D009479:Neuropeptides