Ultrastructural distribution of myosin heavy chain mRNA in cardiac tissue: a comparison of frozen and LR White embedment.
Electron microscopy (EM) in situ hybridization provides the higher resolution necessary to determine the spatial relationship between a specific mRNA and the organelle containing the protein encoded by that message. EM in situ hybridization was used to determine the subcellular myosin heavy chain (MHC) mRNA distribution with respect to the myofibril in normal cardiac tissue. Sections of frozen or acrylic-embedded tissue were compared for ultrastructural integrity and content of endogenous mRNA. Papillary muscles dissected from hearts of normal rabbits were aldehyde-fixed and either frozen or embedded in LR White. EM in situ hybridization with no riboprobe, vector sequence, same-sense, and anti-sense biotinylated riboprobes was detected by indirect immunocytochemistry. Labeling density using an antisense probe was highest over the intermyofibrillar space, with an average signal five times that of background. Background labeling by nonspecific sense probe was consistently low but not random, also having the highest density of gold clusters over the intermyofibrillar space. Ultracryomicrotomy yielded a higher absolute number of gold clusters, but sections were fragmented and disrupted striated muscle morphology. LR White embedment maintained ultrastructural integrity but gave a lower absolute signal. Fortunately, MHC mRNA is an abundant message and can tolerate the decreased sensitivity of LR White.
Wenderoth
M P
MP
Biochemistry Department, University of Washington, Seattle 98195.
Eisenberg
B R
BR
D000180:Acrylic Resins; D000818:Animals; D005615:Freezing; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D009206:Myocardium / Q000737:chemistry / Q000648:ultrastructure*; D009210:Myofibrils / Q000737:chemistry / Q000648:ultrastructure; D009218:Myosins / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D015347:RNA Probes / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis*; D011817:Rabbits; D012518:Sarcomeres / Q000737:chemistry / Q000648:ultrastructure
D000180:Acrylic Resins; D015347:RNA Probes; D012333:RNA, Messenger; C048707:LR white; D009218:Myosins