A rapid method for assessing the distribution of gold labeling on thin sections.
Particulate gold labeling on ultrathin sections is in widespread use for antigen localization at the EM level. To extend the usefulness of gold labeling technology, we are evaluating different methods for sampling and estimating quantities of gold labeling. Here we present a simple, rapid, and unbiased method for assessing the relative pool sizes of immunogold labeling distributed over different cell compartments. The method uses a sampling approach developed for stereology in which a regular array of microscopic fields or linear scans is positioned randomly on labeled sections. From these readouts, gold particles are counted and assigned to identifiable cell structures to construct a gold labeling frequency distribution of those labeled compartments. Here we use ultrathin cryosections labeled for a range of different proteins and for a signaling lipid. We show by scanning labeled sections at the electron microscope that counting 100-200 particles on each of two grids is sufficient to obtain a reproducible and rapid assessment of the pattern of labeling proportions over 10-16 compartments. If more precise estimates of labeling proportions over individual compartments are required (e.g., to achieve coefficients of error of 10-20%), then 100-200 particles need to be counted over each compartment of interest.
Lucocq
John Milton
JM
School of Life Sciences, WTB/MSI Complex, University of Dundee, Dundee DD1 5EH, Scotland, UK. j.m.lucocq@dundee.ac.uk
Habermann
Anja
A
Watt
Stephen
S
Backer
Jonathan M
JM
Mayhew
Terry M
TM
Griffiths
Gareth
G
D000818:Animals; D002460:Cell Line; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008854:Microscopy, Electron; D008867:Microtomy; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods*