Mounting medium

Cultured cells or tissue were fixed with 4% paraformaldehyde for 20 min and washed with PBS three times followed by blocking with 10% normal goat serum plus 1% BSA (Sigma-Aldrich, Shanghai, China) for 45 min, and incubated with rabbit anti-β-catenin (1:100) (Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C in the dark overnight. After washing three times with PBS, cells and tissue were further incubated with cy3-conjugated goat anti-rabbit and HRP-conjugated goat anti-rabbit secondary antibodies for 1 h at 37°C. Subsequently, the slides were cover slipped with mounting medium (Dako, Dalian, Liaoning, China) containing DAPI to counter stain the nuclei and imaged with fluorescence or bright field microscope. Scoring for the slides were performed according to a previous publication [13 ]. Briefly, β-catenin staining with a nuclear localization pattern was examined by two independent pathologists with bright field microscope, and any nuclear staining of breast cancer cells was assumed as positive. Intensity of staining was scored on a scale of 0–3+ as follows: no specific staining (0), weak (1+), moderate (2+), and strong (3+). The Pearson correlation analysis was used to establish the relationship between UCA1 gene expression and nuclear β-catenin levels.

For immunocytochemistry, coverslips with primary neurons were fixed with 4% paraformaldehyde/4% sucrose for 8 min, washed with PBS, and permeabilized with 0.3% Triton-X100/PBS for 10 min. After blocking in 5% NGS/PBS for 30 min, incubation with primary antibodies followed overnight at 4°C: rb-anti-pan-Synapsin (1:500, E028, T. Südhof, Stanford University), ms-anti-PSD-95 (1:500, NeuroMab), ch-anti-MAP2 (1:10,000, Abcam), rb-anti-Cofilin1 (1:7,000, Abcam), rb-anti-protein-interacting-with-C-kinase-1 (PICK1) (1:500, Proteintech), as well as Alexa568-phalloidin (1:100, Invitrogen), all diluted in 5% NGS/PBS. After washing, cells were incubated with the following secondary antibodies: Alexa488 goat-anti-rabbit IgG, Alexa647 goat-anti-chicken IgG (Invitrogen), Cy3-conjugated goat-anti-mouse IgG (Jackson Immuno Research), diluted 1:500 in 5% NGS/PBS for 1 h at RT. After additional washings in PBS, coverslips were embedded in mounting medium (Dako).

Larvae were collected in a cavity block, washed and dissected in 1x PBS. To access the imaginal discs, a cut was made at the posterior two third part of the larvae and slight pressure was applied to expose the contents of the larval gut. The excess tissue was removed and the remaining 1/3rd was turned inside out with the help of a pair of needles. The exposed imaginal discs attached to the inverted cuticle were fixed using 4% paraformaldehyde prepared in 1x PBS containing 0.1% Triton X-100 (1x PBST) for 10 min at room temperature. Discs were given three 1-min rinses with 1x PBST followed by three 10 min washes with the same. Wing discs were blocked for 2 h at room temperature in blocking solution (0.5% BSA, 2% FBS in 1x PBST; pH 7.4). Samples were incubated overnight with primary antibody prepared in blocking buffer minus Triton X-100. Discs were washed twice with blocking buffer and incubated for 2 h at room temperature with fluorescently labeled secondary antibody (Molecular Probes, Invitrogen) prepared in blocking solution without FBS. Two 15 min washes with 1x PBST were performed, followed by two 10 min washes with 1x PBS. Discs were incubated for 10 min with DAPI (SIGMA; working concentration, 1 µg/mL). Two 10-min washes were performed using 1x PBS. The desired imaginal discs were detached and mounted in mounting medium (DAKO-cytomation).

Human VWF was purchased from Merck Chemicals; histamine from Sigma Aldrich, Germany. The recombinant VWF domains (A1, A2, and A3) were produced in HEK293 cells (Posch et al., 2017 (link)). Pneumococcus-specific polyclonal antiserum was generated in rabbit against heat inactivated serotype 35A and 2 bacteria (Pineda, Germany). IgG was purified using protein A/G sepharose. Mouse anti-human VWF antibody (polyclonal IgG) and FITC-conjugated VWF-specific antibody were purchased from Abcam (ab8822), peroxidase-conjugated mouse anti-VWF antibody from Hämochrom Diagnostika; Alexa Fluor 488-conjugated goat anti-rabbit and goat anti-mouse IgG, Alexa Fluor 568-conjugated goat anti-rabbit and goat anti-mouse IgG were from Thermo Fisher Scientific (formerly Invitrogen), paraformaldehyde (PFA) was purchased from EM Science and mounting medium from Dako.