In situ hybridization for the detection of feline interleukin 1 alpha mRNA on the paraffin-embedded section using biotin-labeled probes.
In situ hybridization (ISH) technique with a biotin-labeled probe was established for detecting feline interleukin 1 (IL-1) alpha mRNA in necropsied specimens. Homology between human IL-1 alpha cDNA used as a probe and feline IL-1 alpha mRNA was confirmed by means of dot blot hybridization using the biotin-labeled probe. Hence, we tried by this biotinylated probe to detect mRNA of IL-1 alpha in paraffin-embedded sections. The following results were obtained for the routine procedures: 1) coating slides with poly-L-lysine and/or heating at 60 degrees C at least for 6 hours gave an excellent result for the adhesion of the tissue sections, 2) 10 micrograms/ml solution of proteinase K treatment for 30 minutes or 50 to 100 micrograms/ml solution of proteinase K treatment for 10 to 30 minutes at 37 degrees C gave the good results in the detection of ISH signal, 3) suitable denaturation time of probes at 70 to 90 degrees C was 5 to 15 minutes, and 4) effective hybridization was obtained by incubation for 24 hours at 4 degrees C, for 18 to 24 hours at 25 degrees C or for 5 to 24 hours at 37 degrees C.
Hasegawa
T
T
Department of Veterinary Internal Medicine, Faculty of Agriculture, University of Tokyo, Japan.
Matsumoto
Y
Y
Goitsuka
R
R
Tsujimoto
H
H
Ono
K
K
Hasegawa
A
A
D000818:Animals; D001710:Biotin; D002371:Cat Diseases / Q000276:immunology*; D002415:Cats; D003333:Coronaviridae Infections / Q000276:immunology / Q000662:veterinary*; D004247:DNA / Q000737:chemistry; D007375:Interleukin-1 / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature
D007375:Interleukin-1; D015347:RNA Probes; D012333:RNA, Messenger; D001710:Biotin; D004247:DNA