TY - JOUR
T1 - Immunoelectron microscopy of acetylcholine receptors and 43 KD protein after rapid freezing, freeze-substitution, and low-temperature embedding in Lowicryl K11M
AU - Phillips, G. W.
AU - Bridgman, P. C.
PY - 1991
Y1 - 1991
N2 - To label intracellular determinants of the acetylcholine receptor and associated cytoplasmic proteins while preserving optimal ultrastructure, we developed a post-embedment labeling technique that uses rapid-frozen specimens and freeze-substitution without chemical fixatives. This procedure has been made possible through the use of a low-temperature resin (Lowicryl K11M) that can be polymerized with UV light at -60°C. Rapid-frozen muscle cells were used to evaluate the preservation of structure, and Torpedo electroplaque cells and purified postsynaptic membranes were used to quantitatively evaluate the labeling specificity, efficiency, and resolution of the technique. The labeling efficiency of seven different monoclonal antibodies (MAb) to the acetylcholine receptor varied from 3-13%; there was a correlation between the degree of efficiency and the number of epitopes with which the antibodies reacted. The resolution of the technique was not sufficient to determine whether the antiacetylcholine receptor MAb were bound to the cytoplasmic or the extracellular surface, but was sufficient to correctly determine the location of the receptor-associated 43 KD protein on the cytoplasmic surface.
AB - To label intracellular determinants of the acetylcholine receptor and associated cytoplasmic proteins while preserving optimal ultrastructure, we developed a post-embedment labeling technique that uses rapid-frozen specimens and freeze-substitution without chemical fixatives. This procedure has been made possible through the use of a low-temperature resin (Lowicryl K11M) that can be polymerized with UV light at -60°C. Rapid-frozen muscle cells were used to evaluate the preservation of structure, and Torpedo electroplaque cells and purified postsynaptic membranes were used to quantitatively evaluate the labeling specificity, efficiency, and resolution of the technique. The labeling efficiency of seven different monoclonal antibodies (MAb) to the acetylcholine receptor varied from 3-13%; there was a correlation between the degree of efficiency and the number of epitopes with which the antibodies reacted. The resolution of the technique was not sufficient to determine whether the antiacetylcholine receptor MAb were bound to the cytoplasmic or the extracellular surface, but was sufficient to correctly determine the location of the receptor-associated 43 KD protein on the cytoplasmic surface.
KW - 43 KD protein
KW - Acetylcholine receptors
KW - Colloidal gold
KW - Freeze-substitution
KW - Low-temperature embedding
KW - Post-embedment antibody labeling
KW - Rapid freezing
UR - http://www.scopus.com/inward/record.url?scp=0025798963&partnerID=8YFLogxK
U2 - 10.1177/39.5.2016512
DO - 10.1177/39.5.2016512
M3 - Article
C2 - 2016512
AN - SCOPUS:0025798963
SN - 0022-1554
VL - 39
SP - 625
EP - 634
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
IS - 5
ER -