BRIEF REPORT |
Correspondence to: Torsten Goldmann, Clinical and Experimental Pathology Research Center Borstel, Parkallee 3, D-23845 Borstel, Germany. E-mail: tgoldmann@fz-borstel.de
![]() |
Summary |
---|
![]() ![]() ![]() ![]() |
---|
In primary or cultured cells, in situ hybridization (ISH) or immunocytochemistry (ICC) is often performed on tissue that has been fixed by paraformaldehyde or Carnoy's. Recently we reported an optimized HOPE (HEPESglutamic acid buffer-mediated organic solvent protection effect) fixation protocol for ISH targeting mRNA in lung tissues. We have now examined whether HOPE fixation could also be used on in vitro cultured cells for targeting mRNA by ISH or proteins by ICC on cytospin preparations. Using the myeloid stem cell line KG-1a as a model system, we showed that HOPE fixation can be applied for ISH and ICC on cultured cells. HOPE can be used with cells and tissues and with a broad spectrum of immunohistocytochemical and molecular techniques.
(J Histochem Cytochem 51:977980, 2003)
Key Words: HOPE fixation, paraformaldehyde, Carnoy's solution, cultured cells, ISH, ICC, mRNA, cytokines, cytospins
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() |
---|
HOPE (HEPESglutamic acid buffer-mediated organic solvent protection effect) fixation with subsequent paraffin embedding has recently been described to be a useful new tool for assessment of RNA by in situ hybridization (ISH) in tissues and expanded possibilities for immunohistochemistry (IHC) due to low denaturation of proteins and nucleic acids together with a well-preserved histomorphology (
We first investigated the expression of IL-1ß, GM-CSF, and INF- in KG-1a cells by RT-PCR. Whereas expression of GM-CSF and IL-1ß mRNA was detectable in untreated KG-1a cells (Fig 1A), INF-
expression (Fig 1B) was observed in KG-1a cells incubated with a supernatant of LPS (500 ng/ml, highly purified from Salmonella enterica; Serva, Friedenau, Germany)-stimulated mononuclear cells (SUPLPS) but not in untreated KG-1a cells. Comparison of IL-1ß, GM-CSF, and INF-
expression with GAPDH mRNA expression by quantitative RT-PCR showed 10-fold lower expression of IL-1ß, 100-fold lower expression of GM-CSF (in untreated KG-1a cells), and 20-fold lower expression of INF-
(in SUPLPS-stimulated KG-1a cells) with regard to GAPDH mRNA expression (data not shown).
|
We next examined whether expression of IL-1ß, GM-CSF, and INF- mRNA could also be detected by ISH on cytospin preparations of HOPE-fixed KG-1a cells. To provide at least some comparability to established techniques, we also applied ISH on KG-1a cells fixed with Carnoy's solution or paraformaldehyde (PFA) by targeting INF-
mRNA under the same hybridization conditions used for HOPE fixation, and finally evaluated the results in comparison to HOPE fixation.
For ISH or ICC, KG-1a cells (ATTC No. CCL-246.1; American Type Culture Collection, Rockville, MD) were maintained in Iscove's modified DMEM supplemented with 1% penicillin/streptomycin solution (Gibco/Invitrogen; Karlsruhe, Germany) and 20% heat-inactivated FCS (Linaris; WertheimBettingen, Germany) and were washed several times with PBS. For targeting INF- mRNA by ISH, KG-1a cells were incubated in SUPLPS or medium alone, harvested after 16 hr, and washed several times in PBS. Finally KG-1a cells were resuspended at a concentration of 2 x 106/ml in PBS. KG-1a cells (50,000) were attached to SuperFrost Plus microscope slides (MenzelGläser; Braunschweig, Germany) by centrifugation for 5 min at 450 rpm at high acceleration in a Cytospin 2 centrifuge (Shandon; Frankfurt, Germany) and dried for 10 min at 37C in a sterilizer. After overnight fixation at 4C in HOPE solution (DCS Innovative Diagnostik Systeme; Hamburg, Germany), cells were incubated with acetone/glyoxal for 1 hr at 4C, and dehydrated six times with acetone for 30 min at 4C, followed by two incubations in isopropanol (10 min at 60C, 2 min at 60C) and air-dried. Rehydration was achieved by incubation in 70% (v/v) acetone for 10 min at 4C and DEPC-treated water for 10 min at 4C. Slides were then air-dried. For comparative studies, dried cytospins were fixed for 30 min at 4C in 4% PFA prepared in PBS or for 1 min in Carnoy's solution (60% ethanol, 30% chloroform, and 10% glacial acetic acid) at RT as described elsewhere (
(sense, ATGAAATATACAAGTTATATCTTGGCTTT; antisense, GATGCT-CTTCGACCTCGAAACAGCAT) probes were amplified by PCR for 40 cycles using an AccuPrime Taq DNA Polymerase System (Invitrogen). A 1356-bp fragment obtained by PstI (NEB; Frankfurt am Main, Germany) restriction of pcDNA3 (Invitrogen) was used as a control probe. The probes were labeled overnight with digoxigenin by random primed labeling using High-Prime (Roche; Mannheim, Germany) according to the manufacturer's instructions and labeling efficiency was estimated in comparison to given concentrations of control DNA as described elsewhere (
Whereas no new fuchsin could be detected in KG-1a cells hybridized with no probe (Fig 2A) or the digoxigenin-labeled pcDNA3.1 fragment (Fig 2B), distinct cytoplasmic staining could be observed within 5 min in cells probed with GM-CSF (Fig 2C) or IL-1ß (Fig 2D). Distinct cytoplasmic staining was also detected in SUPLPS-stimulated, HOPE-fixed (Fig 3E) or PFA-fixed KG-1a cells (Fig 3G) probed for INF- mRNA, whereas no staining was detected within 30 min in SUPLPS-stimulated KG-1a cells fixed with Carnoy's solution or in unstimulated KG-1a cells fixed with HOPE, PFA, or Carnoy's solution (Fig 3F; and data not shown).
|
|
In the next group of experiments we tested whether ICC techniques can be also applied to HOPE-fixed KG-1a cells on cytospin preparations and compared the results, first with flow cytometry data of untreated KG-1a cells and second with data obtained from ICC of PFA- and Carnoy-fixed KG-1a cells.
HOPE-, PFA-, and Carnoy-fixed cells on cytospins were prepared as described above and incubated for 60 min with 20 µg/ml of an FITC-conjugated anti-CD34 antibody (Chemicon International; Hofheim, Germany) or an FITC-conjugated anti-CD86 antibody (BD; Heidelberg, Germany) in PBS. CD86 is not expressed on KG-1a cells and was used as an isotype control antibody (Fig 3A). Slides were washed twice with PBS and stained with 10 µg/ml of an Alexa Fluor 488-conjugated goat anti-mouse antibody (Molecular Probes; Leiden, The Netherlands) for 30 min in the dark. After washing twice with PBS, stained cells were mounted with Mowiol (Calbiochem; Schwalbach, Germany) containing 1,4-diazabicyclo[2.2.2]octane (Sigma; Taufkirchen, Germany). Samples were examined with a Leica TCS SP Spectral Confocal Microscope (Leica; Bensheim, Germany) at an excitation wavelength of 488 nm from an argonion laser. For flow cytometry, cultured KG-1a cells were washed with azidePBS containing 10% heat-inactivated human serum (HS), followed by staining for 30 min with 20 µg/ml FITC conjugated anti-CD34 or FITC-conjugated anti-CD86 antibody (BD). Finally, cells were washed twice with azidePBS and analyzed by a FACScalibur (BD) flow cytometer. KG-1a cells expressed high levels of the stem-cell marker CD34, but no CD86 as determined by FACS analysis (Fig 3A). Consistent with FACS data, strong staining for CD34 was observed by ICC in cytospin preparations of HOPE-fixed KG-1a cells (Fig 3C), whereas incubation of HOPE-fixed KG-1a cells with an anti-CD86 antibody resulted in no staining (Fig 3B). Similar results for CD86 staining were observed in PFA- and Carnoy-fixed KG-1a cells (data not shown). Whereas we observed high staining intensities for CD34 in KG-1a cells fixed with HOPE (Fig 3C) or PFA (Fig 3E), only weak staining was detected in KG-1a cells fixed with Carnoy's solution (Fig 3D).
HOPE solution has been shown to be an excellent preservative for human soft tissues, providing protection for proteins and nucleic acids in conjunction with well-preserved morphology. We demonstrated that HOPE is suitable not only for tissue sections but also, with slight modifications, for cultured cells on cytospin preparations.
To prevent osmotic shock, we prepared the hybridization mix used for ISH in KG-1a cells with PBS. Our results indicate that PBS did not affect hybridization specificity because no hybridization signal could be detected in KG-1a cells if ISH was performed with digoxigenin-labeled bacterial DNA. Furthermore, no unspecific signals were observed in untreated KG-1a cells probed for INF-. Because PFA (
mRNA was detected in SUPLPS-stimulated KG-1a cells fixed with Carnoy's solution, comparable signals for INF-
mRNA were detected in HOPE-fixed and PFA-fixed cytospin preparations. In contrast to PFA fixation, KG-1a cells fixed with HOPE or Carnoy's solution retained better morphology.
We could also determine the expression of the cell surface marker CD34 by ICC in HOPE-fixed and PFA-fixed KG-1a cells, whereas in our hands Carnoy-fixed cells showed only weak staining for CD34. This is consistent with findings of other groups showing that Carnoy's fixative reduced the number of chymase-positive cells in ICC staining of mast cells, probably by damaging or changing epitopes, whereas PFA did not (
In conclusion, we showed that both ISH and ICC can be performed in HOPE-fixed cells on cytospin preparations. This may open the opportunity to combine both procedures, providing a powerful tool to better characterize mRNA-expressing cell populations in situ.
![]() |
Acknowledgments |
---|
Supported by the Deutsche Forschungsgemeinschaft (GRK 288, Project A4).
We thank H. Kühl for excellent technical assistance and T. Scholzen and Maria Manoukian (Department of Immunology and Cellular Biology, Research Center Borstel) for confocal microscopy.
Received for publication October 9, 2002; accepted January 16, 2003.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() |
---|
Goldmann T, Suter L, Ribbert D, Otto F (1999) The expression of proteolytic enzymes at the dermal invading front of primary cutaneous melanoma predicts metastasis. Pathol Res Pract 195:171-175[Medline]
Goldmann T, Wiedorn KH, Kuhl H, Olert J, Branscheid D, Pechkovsky D, Zissel G et al. (2002) Assessment of transcriptional gene activity in situ by application of HOPE-fixed, paraffin-embedded tissues. Pathol Res Pract 198:91-95[Medline]
Kanbe N, Kurosawa M, Miyachi Y, Kanbe M, Kempuraj D, Tachimoto H, Saito H (1998) Carnoy's fixative reduces the number of chymase-positive cells in immunocytochemical staining of cord-blood-derived human cultured mast cells. Allergy 53:981-985[Medline]
Kovacs W, Stangl H, Volkl A, Schad A, Dariush FH, Baumgart E (2001) Localization of mRNAs encoding peroxisomal proteins in cell culture by non-radioactive in situ hybridization. Comparison of rat and human hepatoma cells and their responses to two divergent hypolipidemic drugs. Histochem Cell Biol 115:499-508[Medline]
Olert J, Wiedorn KH, Goldmann T, Kuhl H, Mehraein Y, Scherthan H, Niketeghad F et al. (2001) HOPE fixation: a novel fixing method and paraffin-embedding technique for human soft tissues. Pathol Res Pract 197:823-826[Medline]
Pechkovsky DV, Zissel G, Goldmann T, Einhaus M, Taube C, Magnussen H, Schlaak M et al. (2002a) Pattern of NOS2 and NOS3 mRNA expression in human A549 cells and primary cultured AEC II. Am J Physiol 282:L684-692
Pechkovsky DV, Zissel G, Stamme C, Goldmann T, Ari JH, Einhaus M, Taube C et al. (2002b) Human alveolar epithelial cells induce nitric oxide synthase-2 expression in alveolar macrophages. Eur Respir J 19:672-683
(1996) Nonradioactive In Situ Hybridization Application Manual. 2nd ed Mannheim, Germany, Roche Diagnostics, Boehringer Mannheim GmbH
Uhlig U, Haitsma JJ, Goldmann T, Poelma DL, Lachmann B, Uhlig S (2002) Ventilation-induced activation of the mitogen activated proteinkinase pathway. Eur Resp J 20:946-956