Counterstaining improves visualization of the myenteric plexus in immunolabelled whole-mount preparations.
Immunocytochemistry has emerged as a powerful research tool in neurobiology. One of the widely used methods is an indirect fluorescence technique that uses FITC- conjugated IgG to visualise protein expression within tissues, but a major drawback of this technique is the high background fluorescence due to non-specific antibody binding. Gut innervation is complex and best visualized in three-dimensions in whole mount preparations. We describe a simple and easy to use counterstaining procedure in conjunction with an indirect immunofluorescence technique in gut whole mount preparations that largely eliminates background fluorescence and creates a contrasting background against the bright antigen-antibody complexes. Furthermore, this technique allows the detailed qualitative and quantitative study of myenteric plexuses in whole-mount preparations.
Mandhan
Parkash
P
Children's Developmental Genetic Research Group, Department of Surgery, Christchurch School of Medicine and Health Sciences, Christchurch, New Zealand. childoc@msn.com
Qi
Bao Quan
BQ
Keenan
Jacqueline I
JI
Ismail
Salim
S
Beasley
Spencer W
SW
Sullivan
Michael J
MJ
D000818:Animals; D005260:Female; D005333:Fetus / Q000033:anatomy & histology / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods*; D005456:Fluorescent Dyes; D007150:Immunohistochemistry / Q000379:methods; D009197:Myenteric Plexus / Q000033:anatomy & histology* / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000379:methods; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D005456:Fluorescent Dyes; D014660:Vasoactive Intestinal Peptide; D010751:Phosphopyruvate Hydratase