title: An improved, sensitive, non-radioactive in situ hybridization method for the detection of cytokine mRNAs.
abstract: We established an improved non-radioactive in situ hybridization (ISH) method to detect mRNA of cytokines in cell preparations and tissues. Via this method we could demonstrate various cytokines in stimulated peripheral blood mononuclear cells (PBMC), lymphoid cell lines and human lymphoid tissues. The probes for the in situ hybridization were made by labelling cytokine-specific PCR products with digoxigenin (Dig) in a repeated PCR. This resulted in an intrinsic labelling of the probe with several Dig-UTP molecules. Incorporation of Dig-11-dUTPs was shown on ethidium bromide-stained agarose gels by a higher molecular weight of the PCR products with incorporated Dig-dUTPs when compared to control PCR products without digoxigenin. Cytospin-centrifuged cells of PHA-stimulated PBMC or lymphoid cell lines and frozen sections of various human lymphoid tissues were hybridized with the Dig-labelled cytokine probes and the hybridized probes were detected immuno-histochemically. In this way, we detected and localized cytokine mRNAs (IL-2, IL-4, IL-6, IL-8, IL-10) in PBMC, in the human T-cell line Jurkat, in the follicular lymphoma cell line DoHH2, and in human lymph nodes and tonsils. The in situ hybridization had a high sensitivity as the results correlated closely with the detection of cytokine mRNA by reverse transcriptase-PCR (RT-PCR) data from the same samples. We showed that Jurkat and DoHH2 cells produce several cytokines constitutively and that, after activation with the phorbol ester PMA, expression of several cytokine mRNAS was enhanced.
authors.affiliation: Department of Pathology, University Hospital Utrecht, The Netherlands.
authors.forename: S C
authors.initials: SC
authors.lastname: Klein
authors.forename: D F
authors.initials: DF
authors.lastname: van Wichen
authors.forename: J G
authors.initials: JG
authors.lastname: Golverdingen
authors.forename: K C
authors.initials: KC
authors.lastname: Jacobse
authors.forename: R
authors.initials: R
authors.lastname: Broekhuizen
authors.forename: R A
authors.initials: RA
authors.lastname: de Weger
mesh_terms: D001483:Base Sequence; D016207:Cytokines / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004076:Digoxigenin; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D066298:In Vitro Techniques; D007378:Interleukins / Q000235:genetics*; D008221:Lymphoid Tissue / Q000378:metabolism; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D016207:Cytokines; D017931:DNA Primers; D007378:Interleukins; D012333:RNA, Messenger; D013755:Tetradecanoylphorbol Acetate; D004076:Digoxigenin