Subcellular localization of alpha-tubulin and opsin mRNA in the goldfish retina using digoxigenin-labeled cRNA probes detected by alkaline phosphatase and HRP histochemistry.
This paper describes a method for non-radioactive in situ hybridization providing subcellular localization of mRNA in 3 microns cryosections. We used two alternative colorimetric reactions to detect digoxigenin-labeled cRNA probes: alkaline phosphatase and HRP (horseradish peroxidase). With some probes the signal with the alkaline phosphatase reaction was intense, and diffusion of the reaction product was noticeable. Using HRP-conjugated antibodies improved the resolution but decreased the sensitivity of the signal. Photoamplification of the HRP reaction product increased the contrast and improved the sensitivity of the technique.
Barthel
L K
LK
Department of Anatomy and Cell Biology, University of Michigan, Ann Arbor 48109.
Raymond
P A
PA
D000469:Alkaline Phosphatase; D000818:Animals; D004076:Digoxigenin; D006054:Goldfish; D006735:Horseradish Peroxidase; D017403:In Situ Hybridization / Q000379:methods; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D015183:Restriction Mapping; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D017299:Rod Opsins / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014404:Tubulin / Q000096:biosynthesis*
D015347:RNA Probes; D012333:RNA, Messenger; D017299:Rod Opsins; D014404:Tubulin; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase; D004076:Digoxigenin