Axiovision ver 4

GISH has been performed in one diploid hybrid individual B⁶B⁷ (H258) and four allopolyploid individuals, two of AAB⁷B⁷ (H110–1 & 2) and two of B⁶B⁶B⁷B⁷ (H574–1 & 2) composition, using labelled parental diploid genomic DNA as probes [24 (link),29 (link)]. Total genomic DNA from diploid cytotypes AA, B⁶B⁶, and B⁷B⁷ was isolated using the CTAB (Cetyltrimethylammonium Bromide) method [29 (link)] and sheared at 98 °C for 5 min. Approximately 1 μg of genomic DNA of each cytotype was labeled using either digoxigenin or a biotin nick translation kit (Roche). GISH was carried out following the method of [29 (link)].
All preparations after FISH and GISH were analysed with an AxioImager M2 epifluorescent microscope (Carl Zeiss, Vienna, Austria), and images were captured with a CCD camera and processed using AxioVision ver. 4.8 (Carl Zeiss) with only those functions that apply to all pixels of the image equally.

HTM (N25TM-8) cells were plated on glass coverslips and grown to confluency. One week later cells were treated for 4 days with 2μM FUD with or without 2ng/ml TGFβ2 in media containing reduced serum (1%) and no FGF-2. Media was replaced every other day with FUD with or without TGFβ2. After 4 days, cells were fixed with -20˚C methanol for 15 minutes, incubated with a blocking buffer (PBS plus 1% BSA) and then double labeled with anti-fibronectin sera (1:800) and Ist-9 antibody (5μg/ml) or BC-1 antibody (5μg/ml) overnight at 4˚C in blocking buffer. Normal rabbit serum (1:800) and anti-GFAP antibody (5μg/ml) were used as negative controls. Primary antibodies were detected with Alexa 488 goat anti-rabbit or Alexa 546 goat anti-mouse conjugated secondary antibodies (1:500; Thermo Fisher Scientific). Nuclei were labeled with Hoechst 33342. Fluorescence was viewed using an epifluorescence microscope (Axioplan 2; Carl Zeiss Microscopy, LLC) equipped with a digital camera (Axiocam HRm; Carl Zeiss Microscopy LLC) and image acquisition software (Axiovision ver. 4.8, Carl Zeiss Microscopy LLC). All images within an experiment were processed in the exact same way by changing the brightness or contrast to optimize the images.