A quantitative method for assessing co-localization in immunolabeled thin section electron micrographs

Co-localization of glyceraldehyde-3-phosphate dehydrogenase with ferredoxin-NADP reductase in pea leaf chloroplasts

“…1-3). Both subunits are also colocalized with the Calvin cycle enzymes P-glycerate kinase (Anderson et al, 2003), triose-P isomerase, and aldolase (Anderson et al, 2005), but only the B subunit appears to be colocalized with transketolase (Anderson et al, 2006). Both subunits are co-localized with thioredoxin m, but only the A subunit is co-localized with thioredoxin f (Anderson and Carol, unpublished).…”

“…The anti-pea chloroplast GAPD subunit A and subunit B antibodies (provided by Bethyl Laboratories, Montgomery, TX) were raised against peptides representing unique regions of the chloroplast isozymes in sheep and chickens, respectively, and were affinity purified against the immunizing peptides. For details see Anderson et al (2003). Immunoblots of chloroplast proteins probed with the anti-GAPD subunit A and B antibodies have been published.…”

“…Thin sections were cut from pea leaf tissue that had been fixed in 1% acrolein, 0.1% glutaraldehyde and embedded in LR White resin, and were immunolabeled, as described previously (Anderson et al, 1995b(Anderson et al, , 2003. The grids were floated on solution containing both primary antibodies overnight.…”

“…We used the method of Anderson et al (2003) for analysis of nearest neighbor distances on the micrographs from the double-labeling experiments. Briefly, for a population of two different non-interacting species the expression n/N = 1 -exp(−πr 2 ρ) gives the fraction n/N corresponding to position in an ordered list of samples with increasing nearest neighbor distance r, where n is the number of the measurement in rank order, N is the total number of measurements, r is the distance between nearest neighbors, and ρ is the species density (Anderson et al, 2003).…”

“…Briefly, for a population of two different non-interacting species the expression n/N = 1 -exp(−πr 2 ρ) gives the fraction n/N corresponding to position in an ordered list of samples with increasing nearest neighbor distance r, where n is the number of the measurement in rank order, N is the total number of measurements, r is the distance between nearest neighbors, and ρ is the species density (Anderson et al, 2003). A plot of -ln(1 − n/N) versus r 2 produces a straight line, if the two species are distributed randomly.…”