Section V: EM autoradiography

Salpeter, Miriam M.

doi:10.1002/jemt.1060040207

J. Elec. Microsc. Tech.

1986

DOI: 10.1002/jemt.1060040207

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Section V: EM autoradiography

Miriam M. Salpeter

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1987

1988

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Cited by 2 publications

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“…A SEM technique, modified from that of Junger and Bachmann (1980) is described. The procedure had already been illustrated in a nonquantitative manner to study acetylcholine receptors (AChRs) on striated muscle cells in culture (Neugebauer et al, 1985;Salpeter, 1986). In the present study, we calibrated the procedure for sensitivity and chemography and used it to demonstrate gradients of AChRs around the neuromuscular junction (nmj) on innervated and denervated mouse sternomastoid muscle.…”

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Distribution of extrajunctional acetylcholine receptors on a vertebrate muscle: evaluated by using a scanning electron microscope autoradiographic procedure.

Salpeter

Marchaterre

Harris

1988

The Journal of Cell Biology

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Abstract.A scanning electron microscope (SEM) autoradiographic technique was calibrated and used to determine the site density of acetylcholine receptors within 250 ~tm of the neuromuscular junction in innervated as well as 3-and 10-d denervated sternomastoid muscle of the mouse. In all these groups sharp gradients of receptor site density are seen around the endplates in the first 2-7 ~n, continuing less sharply to between 25 and 50 Ixm. Beyond 50 ~tm (to 250 ~tm) a spatial density gradient is present 3 d after denervation, but none exist by 10 d. These results suggest that the postdenervation steady-state extrajunctional receptor site density is reached sooner near the junction than away from the junction.The usefulness of SEM autoradiography to study the expression and distribution of membrane molecules at high resolution is demonstrated.I NFORMATION on the quantitative distribution of cell surface receptors is usually obtained using light microscope or transmission electron microscope (TEM), ~ autoradiography, biochemical-binding studies, and physiological tests. These procedures are limited either by resolution or by an inability to assess variations in receptor distribution over large areas in a morphologically intact cell. In the present study we demonstrate the value of using scanning electron microscope (SEM) autoradiography for that purpose. A SEM technique, modified from that of Junger and Bachmann (1980) is described. The procedure had already been illustrated in a nonquantitative manner to study acetylcholine receptors (AChRs) on striated muscle cells in culture (Neugebauer et al., 1985;Salpeter, 1986). In the present study, we calibrated the procedure for sensitivity and chemography and used it to demonstrate gradients of AChRs around the neuromuscular junction (nmj) on innervated and denervated mouse sternomastoid muscle. The results are consistent with, and extend those previously derived using other techniques.We conclude that SEM autoradiography can be a reproducible quantitative procedure, with sensitivities comparable to those of TEM and light microscope autoradiography and with the potential of answering questions regarding the distribution of surface receptors on a fine structural scale.1. Abbreviations used in this paper: AChR, acetylcholine receptor; nmj, neuromuscular junction; SEM, scanning electron microscope; TEM, transmission electron microscope. Materials and Methods Labeling the ReceptorsThe acetylcholine receptors in the mouse sternomastoid muscle were saturated in vivo by topically applying ~25I-¢t-bungarotoxin onto the surgically exposed muscle in anesthetized mice (10% Nembutal in 10% ethanol, 0.1 cc/10 g body wt) as previously described (Fertuck et al., 1975). Innervated muscles, or muscles denervated either 3 or 10 d previously from adult female mice (25-35 g) were used. Once the receptors on a muscle were saturated, the wound was sutured and the animal allowed to recover for ,o3 h. (We have found that nonspecific binding is more effectively removed in active animals than by ...

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Distribution of extrajunctional acetylcholine receptors on a vertebrate muscle: evaluated by using a scanning electron microscope autoradiographic procedure.

Salpeter

Marchaterre

Harris

1988

The Journal of Cell Biology

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Use of backscattered electron imaging on developed radioautographic emulsions: Application to viewing rat incisor enamel maturation pattern following ⁴⁵calcium injection

McKee

Warshawsky

Nanci

1987

J. Elec. Microsc. Tech.

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Backscattered electron imaging (BED can be used to obtain compositional contrast in biological structures because it detects differences in concentration of elements with different atomic number. Rat incisor enamel shows a banded pattern in the maturation zone when radioautography is used to reveal the location of injected 45Ca at the enamel surface. The bands of developed silver grains in the photographic emulsion that coats the enamel surface are ideal for atomic number contrast. The purpose of this study was to use BE1 to analyze the radioautographic pattern with SEM resolution. Onemonth-old rats were injected with 45Ca and sacrificed at early (1 min, 5 min, 30 min), intermediate (4 h, 8 h), and late (1 day, 4 days) time intervals after injection. Whole incisors were dissected, the enamel organs were removed, and the enamel surface was coated with photographic emulsion and processed for radioautography. These radioautographed teeth were examined with a JEOL JSM-840 SEM equipped with a JEOL backscatter annular-type detector. At the early time intervals, light microscopic examination showed five or six broad black bands running obliquely across the teeth. These were separated by narrow white bands of unlabeled enamel. BE1 resolved the presence of a delicate subbanding pattern within each dark band. At the intermediate time intervals, although the incisal bands persisted, only a diffusely blackened apical area was seen by light microscopy. BE1 resolved bands in this apical region, but these showed no subbanding pattern. At the late time intervals, both light microscopy and BE1 showed no banding, and the enamel was uniformly labeled. The significantly improved resolution obtained with BE1 on surface radioautographs has revealed a previously undetected substructure in the 45Ca-labeled banding pattern seen in enamel maturation.

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Section V: EM autoradiography

Cited by 2 publications

References 41 publications

Distribution of extrajunctional acetylcholine receptors on a vertebrate muscle: evaluated by using a scanning electron microscope autoradiographic procedure.

Distribution of extrajunctional acetylcholine receptors on a vertebrate muscle: evaluated by using a scanning electron microscope autoradiographic procedure.

Use of backscattered electron imaging on developed radioautographic emulsions: Application to viewing rat incisor enamel maturation pattern following ⁴⁵calcium injection

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Section V: EM autoradiography

Cited by 2 publications

References 41 publications

Distribution of extrajunctional acetylcholine receptors on a vertebrate muscle: evaluated by using a scanning electron microscope autoradiographic procedure.

Distribution of extrajunctional acetylcholine receptors on a vertebrate muscle: evaluated by using a scanning electron microscope autoradiographic procedure.

Use of backscattered electron imaging on developed radioautographic emulsions: Application to viewing rat incisor enamel maturation pattern following 45calcium injection

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Use of backscattered electron imaging on developed radioautographic emulsions: Application to viewing rat incisor enamel maturation pattern following ⁴⁵calcium injection