Tierärztliche Hochschule Hannover - TiHo Bibliothek elib
Tierärztliche Hochschule Hannover - TiHo Bibliothek elib Tierärztliche Hochschule Hannover - TiHo Bibliothek elib
carried out by first applying Sudan Black and subsequently cupric sulphate as described above. Photobleaching was performed prior to the removal of paraffin, finally followed by Sudan Black treatment and mounting in glycerol with cover slips. Specific Immunofluorescence Labeling Specific labeling of tissue sections was performed generally as described (Rosenow, et al., 2008; Schnekenburger, et al., 2009) detail, following the pretreatment as indicated, the sections were incubated in PBS containing 5% bovine serum albumin (BSA) for 30 minutes. Primary antibodies were diluted in PBS containing 1% BSA as recommended by the manufacturer (rabbit polyclonal anti-trypsinogen, 1:1000, Abcam, UK; rabbit anti-synaptobrevin2 (VAMP2), 1:100, Wako Chemicals GmbH, Germany; mouse monoclonal anti-Calgranulin B (MRP 1H9), 1:10, Santa Cruz Biotechnology, Germany; chicken anti-Reg3A, raised against a peptide comprising a 16 amino acid N-terminal sequence of human Reg3a, 1:500), incubated for one hour in a wet chamber. The specimen were rinsed repeatedly in PBS to remove excessive antibodies, incubated with Fluorochrome-labeled secondary antibodies (Alexa Fluor® 488 conjugated goat anti-rabbit IgG, 1:500; Alexa Fluor® 488 conjugated goat antichicken IgG, 1:500; Alexa Fluor® 555 conjugated goat anti-rabbit IgG, 1:500; Alexa Fluor® 594 conjugated goat anti-mouse IgG, 1:500; all from Invitrogene, Germany) diluted in PBS containing 1% BSA for one hour, and subsequently rinsed in PBS thoroughly, before mounting as described above. After Sudan Black treatment no detergent should be used during the following procedures since it may destain Sudan Black rapidly (Romijn, et al., 1999). Samples incubated without primary antibodies served as controls for specificity, and indeed did not display detectable immunofluorescence signals. Microscope, Filter Sets and Image Acquisition The sections were examined with an Olympus BX-41 microscope (Olympus Deutschland GmbH, Germany) equipped with filter sets and U-LH100HG for reflected fluorescence microscopy. Objectives used: Olympus PlanN 40x/0.65; Olympus U- Plan FI 100x/1.30 Oil. Epifluorescence filter sets used in this study are shown in Table 1. - 9 -
Tab. 1: Specifications of the filter setups used for fluorescence microscopy Indication Manufacturer Exciter Dichroic mirror Emitter DAPI Semrock*1 set bandpass longpass bandpass filter BFPA-basic 390/18 nm 416 nm 460/ 60 nm exciter, dichroic: FITC filter Olympus*2 U-MNB2 emitter: bandpass 480/20 nm longpass 500 nm ET bandpass 535/ 30 nm Chroma*3 Cy3 filter Olympus set U-MNG 2 bandpass 540/10 nm longpass 570 nm longpass 590 nm Texas Chroma set ET bandpass ET longpass ET bandpass Red filter 49008 560/40 nm 585 nm 630/75 nm *1 Semrock, Rochester, New York, USA; *2 Olympus Deutschland GmbH, Hamburg, Germany; *3 Chroma Technology Corp., Bellows Falls, Vermont, USA All images were acquired with an Olympus digital camera (Olympus U-TV1X-2 CC) using Cell^F imaging-software (Olympus Deutschland GmbH, Germany). The settings for exposure time, contrast, brightness, and pinhole were identical for each set of pictures of a figure, except when indicated otherwise in the text. To allow an improved visibility, images of some figures were adapted by linear brightness enhancement and each of the compared images of the shown figures was treated identically using Cell^F software. Quantification and comparison of fluorescence intensities To quantify fluorescence emission intensities the original acquired image data were used. Mean intensity levels (mean intensity profile, Cell^F imaging software) were calculated from indicated areas of interest, expressed as pixel grey scale values. To compare general autofluorescence of pancreatic tissue samples, mean fluorescence intensities were calculated from selected comparable image areas that display pancreatic acini as the typical histological structure, as shown for example in Fig. 2. - 10 -
- 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 150 und 151: Materials and Methods: Preparation
- 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 -
- Seite 178 und 179: Fig. 6 - 35 -
- Seite 180: Fig. 8 - 37 -
carried out by first applying Sudan Black and subsequently cupric sulphate as<br />
described above. Photobleaching was performed prior to the removal of paraffin,<br />
finally followed by Sudan Black treatment and mounting in glycerol with cover slips.<br />
Specific Immunofluorescence Labeling<br />
Specific labeling of tissue sections was performed generally as described (Rosenow,<br />
et al., 2008; Schnekenburger, et al., 2009) detail, following the pretreatment as<br />
indicated, the sections were incubated in PBS containing 5% bovine serum albumin<br />
(BSA) for 30 minutes. Primary antibodies were diluted in PBS containing 1% BSA as<br />
recommended by the manufacturer (rabbit polyclonal anti-trypsinogen, 1:1000,<br />
Abcam, UK; rabbit anti-synaptobrevin2 (VAMP2), 1:100, Wako Chemicals GmbH,<br />
Germany; mouse monoclonal anti-Calgranulin B (MRP 1H9), 1:10, Santa Cruz<br />
Biotechnology, Germany; chicken anti-Reg3A, raised against a peptide comprising a<br />
16 amino acid N-terminal sequence of human Reg3a, 1:500), incubated for one hour<br />
in a wet chamber. The specimen were rinsed repeatedly in PBS to remove excessive<br />
antibodies, incubated with Fluorochrome-labeled secondary antibodies (Alexa Fluor®<br />
488 conjugated goat anti-rabbit IgG, 1:500; Alexa Fluor® 488 conjugated goat antichicken<br />
IgG, 1:500; Alexa Fluor® 555 conjugated goat anti-rabbit IgG, 1:500; Alexa<br />
Fluor® 594 conjugated goat anti-mouse IgG, 1:500; all from Invitrogene, Germany)<br />
diluted in PBS containing 1% BSA for one hour, and subsequently rinsed in PBS<br />
thoroughly, before mounting as described above. After Sudan Black treatment no<br />
detergent should be used during the following procedures since it may destain Sudan<br />
Black rapidly (Romijn, et al., 1999). Samples incubated without primary antibodies<br />
served as controls for specificity, and indeed did not display detectable<br />
immunofluorescence signals.<br />
Microscope, Filter Sets and Image Acquisition<br />
The sections were examined with an Olympus BX-41 microscope (Olympus<br />
Deutschland GmbH, Germany) equipped with filter sets and U-LH100HG for reflected<br />
fluorescence microscopy. Objectives used: Olympus PlanN 40x/0.65; Olympus U-<br />
Plan FI 100x/1.30 Oil. Epifluorescence filter sets used in this study are shown in<br />
Table 1.<br />
- 9 -
Change language