Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections.
We used a model system to study whether measurements of absolute local antigen concentrations at the electron microscopic level are feasible by counting immunogold labeling density in ultra-thin sections. The model system consisted of a matrix of a variable concentration of gelatin, which was mixed with given concentrations of rat pancreas amylase and fixed according to various fixation protocols. With a relatively mild fixation, there was no clear proportionality between anti-amylase gold labeling and amylase concentration in ultra-thin cryosections. This was presumably due to uncontrolled loss of amylase from the sections. After stronger fixation with 2% glutaraldehyde for 4 hr, labeling density reflected the amylase concentration very well. We observed that matrix (gelatin) density influenced labeling density. A low gelatin concentration of 5% allowed penetration of immunoreagents into the cryosection, resulting in a high and variable labeling density. In gelatin concentrations of 10% and 20%, labeling density was lower but proportional to amylase concentration. To establish an equal (minimal) penetration of immunoreagents, we embedded model blocks with different matrix densities in polyacrylamide (PAA). In ultra-thin cryosections of these PAA-embedded blocks, anti-amylase labeling was proportional to amylase concentration even at a low (5%) gelatin concentration. Anti-amylase labeling in ultra-thin sections from Lowicryl K4M low temperature-embedded blocks was higher than in PAA sections, but the results were less consistent and depended to some extent on matrix density. These results, together with the earlier observation that acrylamide completely penetrates intracellular compartments (Slot JW, Geuze HJ: Biol Cell 44:325, 1982), demonstrate that it is possible to measure true intracellular concentrations of soluble proteins in situ using ultra-thin cryosections of PAA-embedded tissue.
D000180:Acrylic Resins; D000681:Amylases / Q000032:analysis; D000818:Animals; D005629:Frozen Sections; D005780:Gelatin; D006046:Gold; D006651:Histocytochemistry; D007158:Immunologic Techniques; D008297:Male; D008854:Microscopy, Electron / Q000379:methods; D011506:Proteins / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D013205:Staphylococcal Protein A; D000099166:Polyacrylamides
D000180:Acrylic Resins; D000681:Amylases; D005780:Gelatin; D006046:Gold; D011506:Proteins; D013205:Staphylococcal Protein A; D000099166:Polyacrylamides