Eclipse te2000 e confocal laser scanning microscope

Whole brains were dissected from adult fly heads and transferred into 500 μl ice cold PBS. For fixation the brains were then transferred into 4% (w/v) paraformaldehyde in PBS for 1 h at room temperature. Afterwards the fixative was removed and the brains were blocked with 5% normal goat serum in PBS (15 min). The brains were then infiltrated for 2 h with 6E10 (Aβ1-16, monoclonal, mouse, Covance, 1:500), anti-elav (monoclonal, rat, DHSB, 1:50) or anti-myc antibody (ab9106, polyclonal, rabbit, Abcam, 1:500). All antibodies were diluted in PBT (PBS + 0.05% TritonX-100). Unbound antibodies were removed with three washing steps (500 μl PBT, 10 min each) followed by a 1 h incubation with donkey anti-mouse IgG Alexa Fluor®555, donkey anti-rabbit IgG Alexa Fluor®488 (Molecular Probes, 1:200) or anti-rat IgG TRITC (JacksonImmuno Research, 1:200) as appropriate. To remove unbound secondary antibodies, the brains were washed three times with 500 μl PBT, before they were incubated 10 min with 500 μl Hoechst 33342 dye (1 μg/ml in PBS) to stain the nuclei. Finally, the brains were mounted using VECTASHIELD® mounting medium (Vector laboratory). All steps were performed at room temperature, unless indicated otherwise. All samples were analysed using a Nikon ECLIPSE TE2000-E confocal laser-scanning microscope. Pictures were taken at a magnification of 60 ×.

Immunofluorescence was performed for native and transfected cell lines to further confirm CLDN expression. The cells were cultivated on rat collagen type I (Trevigen, Gaithersburg, MD, USA) coated glass coverslips. Thereafter, cells were washed with PBS, fixed wit (1:1) Acetone/Methanol for 5 min at −20 °C and blocked for 30 min with 1% BSA (bovine serum albumin, Sigma-Aldrich, Taufkirchen, Germany) in PBS at 37 °C. CLDNs were stained with primary antibodies (Table 4) diluted in PBS containing 1% BSA, overnight at 4 °C. Cells were washed with PBS. iFlour™ 488 antimouse (AAT Bioquest, Sunnyvale, CA, USA) and iFlour™ 555 antirabbit (AAT Bioquest) were diluted 1:500 in PBS containing 1% BSA and added to the respective cells as secondary antibodies for 1 h at 37 °C. For nuclei staining, DAPI (2 µM) (Sigma-Aldrich) was used. Cells were stored in PBS at 4 °C for further analysis. As a control for unspecific binding sites, cells were also incubated with only the secondary antibodies. Fluorescent images of cells were taken with a Nikon Eclipse TE2000-E confocal laser scanning microscope (400 nm for DAPI, 555 nm for CLDN-3 and -7 proteins, and 488 nm for CLDN-4), with a 60× water immersion objective and software EZ-C1 3.80 (Nikon, Düsseldorf, Germany).

The C. perfringens enterotoxin C-terminal fragment (C-CPE) with an N-terminal Strep-tag II was prepared as described previously [43 (link)]. MDA-MB-231 cell line was used as negative control since it does not express CLDN-3, -4, and -7.
For C-CPE-CLDN binding visualization, the C-CPE was conjugated to green-fluorescent Strep-Tactin^® Chromeo 488 dye (IBA, Goettingen, Germany). The complex was freshly generated before usage, by mixing 2.5 μL Strep-Tactin^® Chromeo 488 (0.5 mg/mL) as recommended by the manufacturer with C-CPE dissolved in elution buffer. The mix was incubated overnight at 4 °C to allow binding of C-CPE with Strep-Tactin^® Chromeo 488. To reach a final concentration of 20 μg/mL C-CPE, the mixture was diluted with 250 µL culture medium. Cells were cultured in a monolayer and stained for 3 h at 37 °C with 20 µg/mL C-CPE-Chromeo 488 complex. For nuclei staining, 1 µM Hoechst 33258 (Sigma-Aldrich) was used. Thereafter, cells were fixed with 4% formaldehyde for 10 min at room temperature and stored in PBS at 4 °C. The cells were imaged with a Nikon Eclipse TE2000-E confocal laser scanning microscope (346 nm for Hoechst 33258 and 488 nm for Chromeo 488) with a 60× water immersion objective and software EZ-C1 3.80 (Nikon).