In situ hybridization using PEG-embedded tissue and riboprobes: increased cellular detail coupled with high sensitivity.
We describe a procedure for preparing tissue sections by embedding in polyethylene glycol for subsequent in situ hybridization analysis using single-stranded RNA probes. Improved tissue morphology is obtained as compared to frozen sections, and the embedding procedure is milder and faster than paraffin embedding. Sections as thin as 2 microns are readily cut from PEG-embedded brain tissue. A simplified hybridization protocol (Clayton et al.: Neuron 1:249, 1988) supports the detection of even low-abundance brain mRNAs (less than or equal to 10(-4) fractional mRNA mass). By employing high stringency washes in place of ribonuclease treatment after hybridization, cell RNA is retained for cresyl violet staining, and high signal:noise ratios are achieved. Solutions to problems with section mounting and adherence to glass slides are presented. The combination of improved morphology, high signal levels, and relative simplicity should make this procedure useful in a variety of applications.
Clayton
D F
DF
Laboratory of Animal Behavior, Rockefeller University, New York, New York 10021.
Alvarez-Buylla
A
A
D000818:Animals; D001345:Autoradiography; D001923:Brain Chemistry; D002171:Canaries; D005629:Frozen Sections; D009693:Nucleic Acid Hybridization; D011092:Polyethylene Glycols; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*
D015347:RNA Probes; D012333:RNA, Messenger; D011092:Polyethylene Glycols