Assessment of transcriptional gene activity in situ by application of HOPE-fixed, paraffin-embedded tissues.
We report the use of HOPE-fixation (HOPE = Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) for specimens utilized for in situ hybridization targeting mRNA. For this purpose, an optimized protocol was developed and repeatedly tested on HOPE-fixed lung specimens. We observed that neither pretreatment, permeabilizing the cells, nor prehybridization is necessary to generate signals. After deparaffinizing, the random primed digoxigenin-labeled probes are directly hybridized together with yeast tRNA for blocking unspecific signals. Detection was performed using anti digoxigenin antibodies conjugated with alkaline phosphatase and new-fuchsine or NBT/BCIP as substrates. The results were verified by RT-PCR and adequate negative controls. Signals for human surfactant protein-A and interferon-gamma-inducible protein-10 developed rapidly within 10 min, accompanied by high signal intensities comparable to those observed in immunohistochemistry. Signal enhancement by biotinyl-tyramide, although giving suitable results as well, did not lead to higher signal intensities, and thus was not necessary in conjunction with the probes tested so far. These experiments were performed with material stored under appropriate conditions (at +4 degrees C) up to five years. To sum up, these initial results, obtained with the novel HOPE-fixative, are promising as regards the enhancement of the capabilities of in situ hybridization in the future.
Goldmann
T
T
Clinical and Experimental Pathology, Research Center Borstel, Germany. tgoldmann@fz-borstel.de
Wiedorn
K H
KH
Kühl
H
H
Olert
J
J
Branscheid
D
D
Pechkovsky
D
D
Zissel
G
G
Galle
J
J
Müller-Quernheim
J
J
Vollmer
E
E
D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008168:Lung / Q000378:metabolism*; D016612:Paraffin Embedding; D011510:Proteolipids / Q000235:genetics / Q000378:metabolism*; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016707:Tissue Fixation / Q000379:methods*; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D011510:Proteolipids; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D012333:RNA, Messenger