In situ hybridization and cytochemistry: localization of mRNA at stained neuromuscular junctions with 33P-labeled probes
| Autor: | Enchi Liu, M. M. Salpeter |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
In situ
Messenger RNA Histology Staining and Labeling Histocytochemistry Neuromuscular Junction In situ hybridization Biology Acetylcholinesterase Molecular biology Staining Mice chemistry.chemical_compound chemistry Cytochemistry Animals Autoradiography RNA Messenger Anatomy Oligonucleotide Probes Phosphorus Radioisotopes In Situ Hybridization |
| Zdroj: | Journal of Histochemistry & Cytochemistry. 42:1407-1411 |
| ISSN: | 1551-5044 0022-1554 |
| DOI: | 10.1177/42.10.7523488 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|