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

1994 ◽  
Vol 42 (10) ◽  
pp. 1407-1411 ◽  
Author(s):  
E Liu ◽  
M M Salpeter
Keyword(s):  
In Situ Hybridization ◽  
Ache Activity ◽  
Neuromuscular Junctions ◽  
Original Method ◽  
Sternocleidomastoid Muscle ◽  
Staining Reaction ◽  
Epsilon Subunit ◽  
Subunit Mrna ◽  
One Step

We modified the Karnovsky 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 Karnovsky and Roots is used to identify the muscle endplates, the stain does not survive the in situ hybridization procedures and association of mRNA to specific endplates can be inferred only indirectly. The successful modification involves secondary staining with diaminobenzidine (DAB) and H2O2 using the Karnovsky-Roots staining reaction product as a catalyst. Mounted longitudinal cryosections of mouse sternocleidomastoid muscle were fixed and stained in one step on the slide with paraformaldehyde plus the Karnovsky-Roots stain, followed by DAB-H2O2 secondary staining. The tissues were then processed for in situ hybridization and probed for the acetylcholine receptor (AChR) epsilon-subunit mRNA, known to be localized at the NMJ. The probe was labeled with 33P, 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 radiolabeling of the AChR epsilon-subunit mRNA were localized at readily identified endplates.

In situ hybridization localization of the Na+ channel β1 subunit mRNA in rat CNS neurons

1994 ◽  
Vol 176 (1) ◽  
pp. 119-122 ◽  
Author(s):  
Youngsuk Oh ◽  
Shunsuke Sashihara ◽  
Stephen G. Waxman
Keyword(s):  
In Situ Hybridization ◽  
Na Channel ◽  
Subunit Mrna ◽  
Cns Neurons ◽  
Rat Cns

scholarly journals Transcripts for the acetylcholine receptor and acetylcholine esterase show distribution differences in cultured chick muscle cells.

1992 ◽  
Vol 118 (5) ◽  
pp. 1201-1212 ◽  
Author(s):  
K W Tsim ◽  
I Greenberg ◽  
M Rimer ◽  
W R Randall ◽  
M M Salpeter
Keyword(s):  
In Situ Hybridization ◽  
Acetylcholine Receptor ◽  
Gaussian Distribution ◽  
Muscle Cells ◽  
Skewed Distribution ◽  
Alpha Subunit ◽  
Subunit Mrna ◽  
Nuclear Label ◽  
The Mean

In situ hybridization of chick cultured muscle cells using exonic DNA probes for both AChR alpha-sub-unit and the catalytic subunit of AChE, revealed major differences in the distribution of label both over nuclei and in their surrounding cytoplasm, although some overlap in these distributions exists. For the AChR alpha-subunit there is a highly skewed distribution of labeled nuclei, with 35% of the nuclei being relatively inactive (less than 0.25 times the mean label) and approximately 10% being very heavily labeled (greater than 2.5 times the mean label). In contrast the nuclei labeled with the exonic probe for the AChE transcripts had a more Gaussian distribution, yet with some slight skewness in the direction of a few heavily labeled nuclei. There was also a difference in the cytoplasmic distribution of the label. The AChR alpha-subunit mRNA was mainly within 4 microns of labeled nuclei while the AChE mRNA was more widely distributed throughout the cytoplasm, possibly within a 10 microns rim around labeled nuclei. An intronic probe for the AChE gave the identical distribution of nuclear label to that of the exonic probe (but without any cytoplasmic label). In addition, calibration of the technique indicated that per myotube the AChE transcript is about sixfold more abundant than the AChR alpha-subunit transcript.

scholarly journals Acetylcholine receptor alpha-subunit mRNA is increased by ascorbic acid in cloned L5 muscle cells: Northern blot analysis and in situ hybridization.

1989 ◽  
Vol 108 (5) ◽  
pp. 1823-1832 ◽  
Author(s):  
O Horovitz ◽  
D Knaack ◽  
T R Podleski ◽  
M M Salpeter
Keyword(s):  
Ascorbic Acid ◽  
In Situ Hybridization ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Alpha Subunit ◽  
Mrna Levels ◽  
Surface Receptors ◽  
Subunit Mrna

Ascorbic acid is the major factor in brain extract responsible for increasing the average acetylcholine receptor (AChR) site density on the cloned muscle cell line L5. In the present study, we show that this effect of ascorbic acid requires mRNA synthesis, and that the mRNA level for the AChR alpha-subunit is increased to about the same level as are the surface receptors. We have found no increase in the mRNA levels of the beta-, gamma-, and delta-subunits, or in the mRNAs of other muscle-specific proteins, such as that of light chain myosin 2, alpha-actin, and creatine kinase. By in situ hybridization, we further show that the increase in alpha-mRNA in response to ascorbic acid is exclusively in myotubes and is located near clusters of nuclei. mRNA levels for the alpha-subunit in mononucleated cells are very low and do not significantly increase in response to ascorbic acid. The mononucleated cells are thus excluded as a possible source for the increase in alpha-subunit mRNA detected by Northern blot analysis. Our results indicate that there is a very specific action of ascorbic acid on the regulation of AChR alpha-mRNA in the L5 muscle cells, and that the expression of surface receptors in these cells is limited by the amount of AChR alpha-subunit mRNA.

Differential expression of human telomerase catalytic subunit mRNA by In situ hybridization in pheochromocytomas

2006 ◽  
Vol 17 (4) ◽  
pp. 387-398 ◽  
Author(s):  
Zuojie Luo ◽  
Jianling Li ◽  
Yinfen Qin ◽  
Yan Ma ◽  
Xinghuan Liang ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Differential Expression ◽  
Catalytic Subunit ◽  
Subunit Mrna ◽  
Human Telomerase

In situ hybridization of CoOX nanoparticles on N-doped graphene through one step mineralization of co-responsive hydrogels

2017 ◽  
Vol 46 (19) ◽  
pp. 6163-6167 ◽  
Author(s):  
Jing Ma ◽  
Xiaojuan Wang ◽  
Ting He ◽  
Minli Tan ◽  
Jun Zheng ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Electrocatalytic Properties ◽  
Doped Graphene ◽  
One Step ◽  
Responsive Hydrogels

Involving GO into Co-PT hydrogels can enhance the strength of hydrogels. The electrocatalytic properties of CoOX/N-rGO700 aerogels are improved by annealing.

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