Resolution in EM autoradiography V: 45Ca (and 33P).

Salpeter, Miriam M.; Nelson, Rob; Harris, Rosemarie; Salpeter, E. E.

doi:10.1177/35.10.3624849

Cited by 6 publications

(5 citation statements)

References 9 publications

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“…We successfully used 33P-labeled probes confirming earlier studies (3,7,10,11) which predicted that 33P would be a usable isotope for in situ hybridization. 33P is preferable to 3% for in situ hybridization because it eliminates one potential problem of background due to disulfide bonding with nonspecific sites on the tissue.…”

Section: Use Of 33p For In Situ Hybridizationsupporting

confidence: 83%

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In situ hybridization and cytochemistry: localization of mRNA at stained neuromuscular junctions with 33P-labeled probes.

Liu¹,

Salpeter²

1994

J Histochem Cytochem.

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We modified the Kamovsky and Roots method of staining sites of acetylcholinesterase (AChE) activity at neuromuscular junctions (NMJs) to survive the lengthy, multiple steps of in situ hybridization and autoradiography. When the original method of Kamovsky and Roots is used to identify the musde endplates, the stain does not survive the in situ hybridization procedures and association of mRNA to specific endplates can be iderred only indirectly. The successful modification involves secondary staining with diaminobenzidine (DAB) and H202 using the Kammkj-hts staining reaction product as a catalyst. Mounted longitudiaal agosections of mouse stemodeidomastoid musde were fd and stained in one step on the slide with paraformaldehyde plus the Kamovsky-Roots stain, followed by DAB-H202 secondary staining. The tissues were then p d for in situ hybridization and probed for the acetylcholine receptor (AChR) &-subunit "A, known to be localized at the NMJ.The probe was labeled with 33pI which is ideal for in situ hybridization. By this procedure, the endplate stain was retained even after the hybridization and autoradiographic procedures, and the developed grains due to radiolabehg of the AChR &subunit mRNA were localized at readily identified endplates. ( J Histochem Cytocbem 42:1407-1411,

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Section: Use Of 33p For In Situ Hybridizationsupporting

confidence: 83%

“…In addition, the energy of 33P (0.26 MeV), which is comparable to that of 45Ca, is only a factor of 1.5 higher than that of 35S (0.17 MeV) or 14C (0.16 MeV). The autoradiographic sensitivity (3) and resolution (11,12) to 33P are therefore not much different from those with 31S (14C).…”

Section: Use Of 33p For In Situ Hybridizationmentioning

confidence: 95%

In situ hybridization and cytochemistry: localization of mRNA at stained neuromuscular junctions with 33P-labeled probes.

Liu¹,

Salpeter²

1994

J Histochem Cytochem.

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“…The fine structural resolution will of course be better with the TEM. Since geometric factors are the main cause for poor resolution when higher energy isotopes are used (Salpeter et al, 1977(Salpeter et al, , 1988, resolution should be excellent with surface receptors labeled with any isotope because they represent a very thin specimen. The resolution would then be limited only by the emulsion thickness, the size of the silver halide crystal of the emulsion, and the size of the developed grain which have all been established for L4 (Ilford) and D-19 development (Salpeter et al, 1977).…”

Section: Resolutionmentioning

confidence: 99%

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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“…To determine the grain distribution, a 275nm-radius circle is centered over each grain . Our choice of this size circle is based on both theoretical and experimental data showing that 275 nm approximates the half distance for a point source of calcium (Salpeter et al, 1987;Winegrad, Fig. 2. A tangential section was cut through renal artery cells after incubation with 45Ca, freeze drying, and preparation for EM ARG.…”

Section: Data Collection and Analysismentioning

confidence: 99%

Effects of temperature and buffer composition on calcium sequestration by sarcoplasmic reticulum and plasma membrane of rabbit renal artery

et al. 1990

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45Ca electron microscopic autoradiography was used to examine the effects of buffer composition and temperature on the distribution of calcium in rabbit renal artery smooth muscle cells. The results show that the relative distribution of calcium is dependent on both the buffer used (Tris or Krebs) and the temperature of the bathing solution (25 degrees C or 34 degrees C). Krebs buffer at 34 degrees C gave the highest relative activity in the plasma membrane, sarcoplasmic reticulum, and mitochondria. Buffer and temperature had little effect on the relative activity of the nucleus or cytoplasm. Next, we identified the cellular sites of calcium accumulation after 5, 15, 30, or 60 min exposure to 45Ca in Krebs buffer at 34 degrees C. The results show that sarcoplasmic reticulum and plasma membrane are the primary sites of calcium accumulation during influx into these cells. Although the amount of 45Ca in the cell continues to increase with longer exposure, the relative distribution of calcium is essentially the same after 5 or 60 min. The data also indicate that the relative activity of plasma membrane + sarcoplasmic reticulum (a combination site that includes sarcoplasmic reticulum within a mean distance of 275 nm of the plasma membrane) is similar to the membrane alone and is lower than the sarcoplasmic reticulum alone.

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Resolution in EM autoradiography V: 45Ca (and 33P).

Cited by 6 publications

References 9 publications

In situ hybridization and cytochemistry: localization of mRNA at stained neuromuscular junctions with 33P-labeled probes.

In situ hybridization and cytochemistry: localization of mRNA at stained neuromuscular junctions with 33P-labeled probes.

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

Effects of temperature and buffer composition on calcium sequestration by sarcoplasmic reticulum and plasma membrane of rabbit renal artery

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