Applications of an efficient method for comparing immunogold labelling patterns in the same sets of compartments in different groups of cells.
Quantitative immunoelectron microscopy often involves determining the distributions of gold label in different intracellular compartments and then drawing comparisons between compartments in the same sample of cells or between experimental groups of cells. In the case of within-group comparisons, recent developments in the estimation of relative labelling index and labelling density make it possible to test whether or not particular compartments are preferentially labelled. These methods are ideally suited to analysing gold label restricted to volume (organelle) or surface (membrane) compartments but may be modified to analyse label localised in mixtures of both. Here, a simple and efficient approach to drawing between-group comparisons for label associated with organelles and/or membranes is presented. The method relies on multistage random sampling of specimens (via blocks and microscopic fields) followed by simply counting gold particles associated with different compartments. The distributions of raw gold counts in different groups are then compared by contingency table analysis with statistical degrees of freedom for chi-squared values being determined by the number of compartments and the number of experimental groups of cells. Compartmental chi-squared values making substantial contributions to the total chi-squared values then identify where the main between-group differences reside. The method requires no information about compartment size (for example, organelle profile area or membrane trace length) and does not even depend critically on standardising between-group magnification. Its application is illustrated using datasets from immunolabelling studies designed to localise the KDEL receptor, phosphatidyl-inositol 4,5-bisphosphate, GLUT4 and rab4 at the electron microscopic level.
Mayhew
Terry M
TM
Centre for Integrated Systems Biology and Medicine, Institute of Clinical Research and School of Biomedical Sciences, Queen's Medical Centre, E Floor, University of Nottingham, NG7 2UH, Nottingham, UK. terry.mayhew@nottingham.ac.uk
Griffiths
Gareth
G
Lucocq
John M
JM
D051275:Glucose Transporter Type 4; D017979:Gold Colloid; D006056:Golgi Apparatus / Q000502:physiology / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D046529:Microscopy, Electron, Transmission; D016253:Microscopy, Immunoelectron; D009004:Monosaccharide Transport Proteins / Q000737:chemistry; D009124:Muscle Proteins / Q000737:chemistry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry; D018000:Receptors, Peptide / Q000737:chemistry; D013347:Subcellular Fractions / Q000502:physiology / Q000648:ultrastructure; D016707:Tissue Fixation; D020695:rab4 GTP-Binding Proteins / Q000737:chemistry
D051275:Glucose Transporter Type 4; D017979:Gold Colloid; C064459:KDEL receptor; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D018000:Receptors, Peptide; C495692:SLC2A4 protein, human; D020695:rab4 GTP-Binding Proteins