A simple method for comparing immunogold distributions in two or more experimental groups illustrated using GLUT1 labelling of isolated trophoblast cells.
Colloidal gold-labelling, combined with transmission electron microscopy, is a valuable technique for high-resolution immunolocalization of identified antigens in different subcellular compartments. Whilst the technique has been applied to placental tissues, few quantitative studies have been made. Subcellular compartments exist in three main categories (viz. organelles, membranes, filaments/tubules) and this affects the possibilities for quantification. Generally, gold particles are counted in order to compare either (a) compartments within an experimental group or (b) compartmental labelling distributions between groups. For the former, recent developments make it possible to test whether or not there is differential (nonrandom) labelling of compartments. The methods (relative labelling index and labelling density) are ideally suited to analysing label in one category of compartment (organelle or membrane or filament) but may be adapted to deal with a mixture of categories. They also require information about compartment size (e.g. profile area or trace length). Here, a simple and efficient method for drawing between-group comparisons of labelling distributions is presented. The method does not require information about compartment size or specimen magnification. It relies on multistage random sampling of specimens and unbiased counting of gold particles associated with different compartments. Distributions of observed gold counts in different experimental groups are compared by contingency table analysis with degrees of freedom for chi-squared (chi(2)) values being determined by the numbers of compartments and experimental groups. Compartmental values of chi(2)which contribute substantially to total chi(2)identify the principal subcellular sites of between-group differences. The method is illustrated using datasets from immunolabelling studies on the localization of GLUT1 glucose transporters in cultured human trophoblast cells exposed to different treatments.
Mayhew
T M
TM
Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, E Floor, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK. terry.mayhew@nottingham.ac.uk
Desoye
G
G
D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D005260:Female; D005947:Glucose / Q000008:administration & dosage; D051272:Glucose Transporter Type 1; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008854:Microscopy, Electron; D009004:Monosaccharide Transport Proteins / Q000032:analysis*; D011247:Pregnancy; D014327:Trophoblasts / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure
D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human; D005947:Glucose