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Summary 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 °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.

We have developed a novel method for tissue fixation, including subsequent paraffin-embedding and sectioning, that allows the complete pathological analysis of all types of human soft tissues. Furthermore, it maintains additional positive features relevant to immunohistochemistry and molecular pathology. The so-called HOPE-technique (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) comprises a protection-solution with an organic buffer, acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature. Although the exact mechanism of protection has still to be elucidated, it seems rather unlikely that chemical bindings occur during the whole process of fixation, which is described and compared with the standard formalin-paraffin technique. Essentially, HOPE-fixed sections show formalin-like morphology. However, the sections are somewhat difficult to handle because of their fragility. This is due to the absence of any type of protein cross-linking and the dynamic processes of immersion and outflow of the HOPE protection solution. HOPE-fixed sections provide an excellent preservation of proteins and antigenic structures for differential analysis by immunohistochemical and/or enzyme histochemical techniques. However, their most remarkable feature is the extremely low degradation of nucleic acids (DNA and RNA) combined with good results obtained by in situ hybridization techniques. In conclusion, HOPE fixation may become a valuable additional tool in modern pathology.

In severe pneumonia, the application of granulocyte-colony stimulating factor (G-CSF) was associated with reduced complications possibly by an induction of anti-inflammatory cytokines. It is not clear, whether G-CSF induces interleukin-10 (IL-10) synthesis in neutrophils. In a randomized study, 15 patients with severe community acquired pneumonia were treated either by a single dose of G-CSF and antibiotic therapy (n=8) or antibiotics alone (n=7). Messenger ribonucleic acid (mRNA) expression of IL-10 and tumor necrosis factor alpha of peripheral blood leukocytes was measured using in-situ hybridization (ISH) and reverse-transcription-polymerase-chain-reaction (RT-PCR). In addition, the cytokine release of lipopolysaccharide (LPS)-stimulated whole blood was measured by ELISA. We detected increased IL-10 mRNA by ISH (140 +/- 8% vs. -11 +/- 5%, P0.01) and RT-PCR (126 +/- 16% vs. -28 +/- 3%, P0.01) in the G-CSF-treated group only. In contrast, LPS-stimulated whole blood cells in vitro released significantly less IL-10 compared to the control group (-38.2 +/- 97 vs. -14.8 +/- 6 pg/ml, P0.02). There was no significant effect on IL-10 serum protein levels and the TNF-alpha release and expression. IL-10 mRNA was detected predominantly in cluster designation 66b (CD66b) positive nucleated blood cells indicating that polymorphonuclear leukocytes are the main source of IL-10 expression after G-CSF stimulation. G-CSF induces transcription of IL-10 mRNA in neutrophils without increased release. This may be due to posttranscriptional effects.

One hundred paraffin-embedded cervical biopsy specimens were tested for the presence of human papilloma virus (HPV) by in situ hybridization (ISH), and by direct and indirect in situ PCR (IS-PCR) in order to evaluate the efficiency of the different in situ methods in detecting HPV infection. ISH was performed using either commercial DNA probes or a cocktail of 5′-digoxigenin labeled oligoprimers. The same were used for ISH during indirect IS-PCR. To enhance the sensitivity of ISH several polymers, i.e., polyvinyl alcohol (PVA), polyethylene glycol, and polyvinylpyrrolidone were added to the alkaline phosphatase nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) reaction. Furthermore, tyramide signal amplification (TSA) was tried for signal amplification. Those samples treated with PVA during the NBT/BCIP reaction did not show any signal amplification whereas those treated with TSA exhibited a dramatic increase in sensitivity with usually acceptable signal to noise ratios. Our results show that, regarding sensitivity, ISH with subsequent signal amplification by TSA can be used as an almost equivalent alternative to the more cumbersome IS-PCR on routinely processed tissue specimens. When considering reproducibility, it is superior to IS-PCR.