An in situ hybridization technique to detect low-abundance slug mRNA in adherent cultured cells.
In this chapter, we describe a simple and relatively rapid technique for detecting low-abundance slug mRNA in cultured cells. The procedure uses nonradioactive digoxigenin-labeled RNA probes that are more sensitive than deoxyribonucleic probes and simpler to detect than radioactively labeled probes. Cells are grown in glass chamber slides, fixed in an acidic fixative, dehydrated through ethanol and xylene, permeabilized in pepsin, and post-fixed. Slides are then incubated overnight at 37 degrees C in a buffer containing 50% formamide and 5-10 ng/gL probe. Excess probe is removed by washing at high temperature in low-salt buffer and by treatment with RNase. Probe is detected immunohistochemically with an anti-digoxigenin Fab fragment, using a tyramide amplification kit to enhance signal and Fast Red for visualization. The technique has the advantages of probe stability and sensitivity, hybridization at low temperature, rapidity and sensitivity of probe detection, and the production of permanent specimens.
Choi
Changsun
C
Department of Veterinary Biosciences, Ohio State University, Columbus, Ohio, USA.
Hudson
Laurie G
LG
Savagner
Pierre
P
Kusewitt
Donna F
DF
D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002460:Cell Line; D004076:Digoxigenin / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D015603:Keratinocytes / Q000502:physiology; D015347:RNA Probes / Q000138:chemical synthesis; D012333:RNA, Messenger / Q000032:analysis*; D012680:Sensitivity and Specificity; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology
D015347:RNA Probes; D012333:RNA, Messenger; C000604407:SNAI1 protein, human; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; D004076:Digoxigenin