Tierärztliche Hochschule Hannover - TiHo Bibliothek elib
Tierärztliche Hochschule Hannover - TiHo Bibliothek elib Tierärztliche Hochschule Hannover - TiHo Bibliothek elib
Materials and Methods: Preparation of Tissue Sections: Tissue samples were essentially prepared as previously described (Schnekenburger, et al., 2008). Samples derived from biopsies taken in the course of surgeries were fixed with buffered formaldehyde solution, washed and embedded in paraffin for archival storage. Samples from paraffin-blocks were cut into 4-µm-thick sections (HM315 Microtom, Microm international GmbH, Germany), mounted on polylysinecoated microscope slides (Menzel, Germany), thoroughly air-dried and stored until use as previously described (Mayerle, et al., 2005). Paraffin was removed from sections, by immersion three times for 10 minutes in 98.5% isomeric xylene (Carl Roth, Germany). To rehydrate the tissue, slides were progressively incubated in decreasing concentrations of graded ethanol (5 min 100%, 5 min 96%, and 5 min 70%), washed in deionized water and transferred to phosphate buffered saline (PBS; 137 mM sodium chloride, 81 mM di-sodium hydrogenphosphate, 27 mM potassium chloride, 15 mM potassium dihydrogenphosphate, pH 7.2 - 7.4). To allow for retrieval of antigenic sites, the sections were immersed in Tris/EDTA retrieval buffer at pH 9 (EnVision FLEX Target Retrieval Solution, high pH, DAKO, Denmark) and heated in a steamer (BraunMultiGourmet FS20, Braun GmbH, Germany) for 35 minutes. The temperature was then adjusted to room temperature and the slides transferred into PBS. Sudan Black Treatment A fresh, saturated solution of Sudan Black B (Sudanschwarz B C.I. 26150, Carl Roth, Germany) was prepared (0.25% (w/v) Sudan Black B in 70% isopropyl alcohol) and stirred over night at room temperature. Non-solubilized dye particles were precipitated by centrifugation at 3000xg for 20 minutes. The supernatant was filtered (595½ filter, Whatman® Schleicher and Schuell, Germany) and stored air-tight sealed in the dark until usage. Solutions older than 3 to 4 weeks were discarded to avoid accumulation of polyazo derivates that may form during prolonged storage (Horobin RW, 2002). Tissue sections on slides were transferred into the Sudan Black solution and incubated air-tight sealed for indicated periods in the dark (incubation time of 90 minutes was used as a standard protocol). Afterwards, specimen were - 7 -
once briefly dipped in 70% isopropyl alcohol, to avoid precipitation of Sudan Black grains. Excessive Sudan Black was wiped off manually from the back and along the edges of the slides with non-pilling tissues. Before mounting or specific immunolabeling, slides were rinsed four times briefly in PBS. All samples shown were embedded in glycerol without supplements. However, in order to reduce photobleaching effects during prolonged light exposition, we recommend the use of 8% (w/v) n-propyl gallate (Sigma-Aldrich, Germany), dissolved in 120 mM sodium acetate (pH 7.2), 90% glycerol. Aliquots of this embedding medium can be stored in the dark, at -20ºC for several months, or kept at 4ºC for not more than one to two weeks, until usage. Cupric Sulphate Treatment The slides with sections were immersed in 10, 20, 50, or 100 mM of cupric sulphate (copper II sulphate pentahydrate, Sigma-Aldrich, Germany) solved in 50 mM ammonium acetate, pH 5.0, for 60 to 90 minutes in the dark and were washed in PBS before mounting (Schnell, et al., 1999). Fresh cupric sulphate solution was prepared before each experiment. Toluidine Blue Treatment Tissue sections were stained for 8 hours with 0.1% (w/v) Toluidine Blue O (Carl Roth, Germany) solution which was freshly prepared in 70% isopropyl alcohol. Afterwards slides were briefly rinsed in 70% isopropyl alcohol and washed in PBS. Photobleaching Irradiation of the tissue sections was performed before removal of paraffin on a 302 nm UV transilluminator (model IL350M, 180 W, Bachofer, Germany). For this purpose, slides were positioned for 2 hours with the tissue sections upside-down facing the transilluminator screen, without any cover and keeping a distance of 4 mm to the screen surface to avoid excessive heating of the specimen. Combination of Sudan Black with other Methods For combined Sudan Black and Toluidine Blue treatment, 0.1% (w/v) Toluidine Blue O was dissolved in a saturated Sudan Black B solution and tissue slides were incubated for 90 minutes. Treatment with cupric sulphate and Sudan Black was - 8 -
- Seite 100 und 101: 5.4 Ergebnisse der quantitativen Pr
- Seite 102 und 103: Abb. 19: ROC-Analyse der S100A8/A9
- Seite 104 und 105: signifikant, während sich die Grup
- Seite 106 und 107: Abb. 22: ROC-Analyse der Plasmakonz
- Seite 108 und 109: 6. Diskussion Das Pankreas produzie
- Seite 110 und 111: Das zweite Protein, das in dieser A
- Seite 112 und 113: ICAM-1 des Endothels erhöht. Fasst
- Seite 114 und 115: (Ghavami 2004) beschrieb die Indukt
- Seite 116 und 117: Adenokarzinom meistens im Pankreask
- Seite 118 und 119: Reg3A für die Aggregation von Bakt
- Seite 120 und 121: homogener Tumortypen zur Immunfluor
- Seite 122 und 123: spricht dafür, dass man chronische
- Seite 124 und 125: Als Indikator der Überexpression v
- Seite 126 und 127: detektiert werden. In der Mehrzahl
- Seite 128 und 129: 8. Summary Till Erben: “Detection
- Seite 130 und 131: ER Fab Fc GAPDH (G/V) h HE HPF HRP
- Seite 132 und 133: Puffer RNA ROC ROS rpm RT SB Sens.
- Seite 134 und 135: Chelvanayagam DK, Beazley LD (1997)
- Seite 136 und 137: Keim V, Iovanna JL, Rohr G, Usadel
- Seite 138 und 139: Ni XG, Bai XF, Mao YL, et al. (2005
- Seite 140 und 141: Strobel O, Rosow DE, Rakhlin EY, et
- Seite 142 und 143: 11. Danksagung Mein herzlicher Dank
- Seite 144 und 145: What to do with high autofluorescen
- Seite 146 und 147: Introduction: The preservation of t
- Seite 148 und 149: erythrocytes (Baschong, et al., 200
- Seite 152 und 153: carried out by first applying Sudan
- Seite 154 und 155: For each set of compared experiment
- Seite 156 und 157: was visualized using bright field m
- Seite 158 und 159: prior to the application immunolabe
- Seite 160 und 161: Discussion: Intrinsic and induced a
- Seite 162 und 163: effects on the fluorescent staining
- Seite 164 und 165: Toluidine Blue has been described t
- Seite 166 und 167: References: Andersson, H., Baechi,
- Seite 168 und 169: Rosenow, F., Ossig, R., Thormeyer,
- Seite 170 und 171: Tab. 1: Specifications of the filte
- Seite 172 und 173: Figure legends Fig. 1 Localization
- Seite 174 und 175: conjugated antibodies reveal mature
- Seite 176 und 177: Fig. 2 Fig. 3 - 33 -
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Materials and Methods:<br />
Preparation of Tissue Sections:<br />
Tissue samples were essentially prepared as previously described (Schnekenburger,<br />
et al., 2008). Samples derived from biopsies taken in the course of surgeries were<br />
fixed with buffered formaldehyde solution, washed and embedded in paraffin for<br />
archival storage. Samples from paraffin-blocks were cut into 4-µm-thick sections<br />
(HM315 Microtom, Microm international GmbH, Germany), mounted on polylysinecoated<br />
microscope slides (Menzel, Germany), thoroughly air-dried and stored until<br />
use as previously described (Mayerle, et al., 2005).<br />
Paraffin was removed from sections, by immersion three times for 10 minutes in<br />
98.5% isomeric xylene (Carl Roth, Germany). To rehydrate the tissue, slides were<br />
progressively incubated in decreasing concentrations of graded ethanol (5 min 100%,<br />
5 min 96%, and 5 min 70%), washed in deionized water and transferred to phosphate<br />
buffered saline (PBS; 137 mM sodium chloride, 81 mM di-sodium<br />
hydrogenphosphate, 27 mM potassium chloride, 15 mM potassium dihydrogenphosphate,<br />
pH 7.2 - 7.4). To allow for retrieval of antigenic sites, the<br />
sections were immersed in Tris/EDTA retrieval buffer at pH 9 (EnVision FLEX<br />
Target Retrieval Solution, high pH, DAKO, Denmark) and heated in a steamer<br />
(BraunMultiGourmet FS20, Braun GmbH, Germany) for 35 minutes. The temperature<br />
was then adjusted to room temperature and the slides transferred into PBS.<br />
Sudan Black Treatment<br />
A fresh, saturated solution of Sudan Black B (Sudanschwarz B C.I. 26150, Carl Roth,<br />
Germany) was prepared (0.25% (w/v) Sudan Black B in 70% isopropyl alcohol) and<br />
stirred over night at room temperature. Non-solubilized dye particles were<br />
precipitated by centrifugation at 3000xg for 20 minutes. The supernatant was filtered<br />
(595½ filter, Whatman® Schleicher and Schuell, Germany) and stored air-tight<br />
sealed in the dark until usage. Solutions older than 3 to 4 weeks were discarded to<br />
avoid accumulation of polyazo derivates that may form during prolonged storage<br />
(Horobin RW, 2002). Tissue sections on slides were transferred into the Sudan Black<br />
solution and incubated air-tight sealed for indicated periods in the dark (incubation<br />
time of 90 minutes was used as a standard protocol). Afterwards, specimen were<br />
- 7 -
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