4 6 diamidino 2 phenylindole dapi containing mounting medium

ICC was performed as we have recently described [17 (link)]. SVGA cells were infected with wild-type Mad-1 JCV and transfected twice with expression plasmid for FLAG-NEMO on days 2 and 5 postinfection. Cells were fixed in 4% paraformaldehyde, in PBS for 10 min, washed, permeabilized for 5 min with 0.1% Triton X-100, blocked with 5% normal goat serum for 30 min and incubated for 3 h at 37°C with mouse anti-FLAG or mouse anti-VP1 antibody at a 1:100 dilution in PBS. Cells were then washed, incubated for 2 h with secondary FITC-conjugated goat anti-mouse secondary antibody at a 1:200 dilution, washed, mounted with 4',6-diamidino-2-phenylindole (DAPI)-containing mounting medium (VECTASHIELD, Vector Laboratories Inc. Burlingame, CA), and viewed by fluorescence microscopy.

EHM rings or fibers were washed in PBS (Thermo Fisher), fixed in 4% paraformaldehyde (Klinipath) for 20 min, stained in eosin (J.T.Baker) for 1 h and stored overnight in 70% ethanol (Sigma Aldrich). Finally, the EHM ring or fiber was embedded into paraffin wax. Sections of 5 µm were cut using a rotary microtome.
Hematoxylin and eosin (H&E) staining was performed to gain insight in the cellular distribution and the extracellular matrix (ECM) structure. After rehydration, the slides were placed in Hematoxylin (5 min), washed with running tap water (10 min), placed in eosin (1 min) and washed with demi water. Dehydration steps were performed and the slides were closed with Entallan. Images were obtained using a Leica Microscope (5×, 10× or 20× magnification).
Vimentin and CNA35 IHC were performed to visualize vimentin-positive cells, implicated on being CF and/or collagen matrix. Slides were stained with vimentin antibody (1/150 dilution, ab92547, Abcam, Cambridge, UK) followed by appropriate secondary antibody (1/500 dilution) or adding CNA35 (1/100 dilution) [40 (link),41 (link)]. Sections were further incubated in 4′,6′-diamidino-2-phenylindole (DAPI)-containing mounting medium (Vector Laboratories, Burlingame, USA) to stain nuclei. Images were obtained using a Leica fluorescent microscope (40× magnification) or a Leica SPE confocal microscope (63× magnification).

Skin sections were deparaffinized in xylene and rehydrated in a graded ethanol series. After heat-induced epitope retrieval in citrate buffer, pH 6.0 (Scytek Laboratories, Inc.), sections were incubated with 3% bovine serum albumin blocking reagent for 10 min at room temperature. After blocking, sections were incubated with a primary antibody against VEGF (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) followed by incubation with Alexa Fluor 488 anti-rabbit secondary antibody (Biolegend, San Diego, CA, USA). The sections were mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting medium (Vector Laboratories Inc., Burlingame, CA, USA) and observed under an inverted microscope (Axio Observer 7; Carl Zeiss Microscopy GmbH). VEGF-positive cells were counted manually in five microscopic fields in each stained sample, and the mean value was used for statistical analyses.

WT and VPS35^D620N SH-SY5Y cells that were seeded on glass coverslips in 24-well plates were fixed in 4% paraformaldehyde in PBS for 10 min at RT. Cells were then permeabilized in 0.1% Triton X-100 in PBS for 10 min and blocked with 5% donkey serum (Abcam) in PBS for 1 h. The coverslips were then incubated overnight at 4 °C with the primary antibodies diluted in blocking buffer and for 1 h at RT for secondary antibody incubation. Coverslips were finally mounted onto glass slides in 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting medium (Vector Laboratories, Burlingame, CA). The slides were analyzed using either structured illumination microscopy (SIM) or confocal microscopy. SIM images were acquired with an AxioObserver Z1 compound microscope (Carl Zeiss, Oberkochen, Germany) equipped with an Apotome, 63x oil objective and an AxioCam MRm3 CCD camera (Carl Zeiss). Confocal images were acquired with a TCS SP8 high-resolution confocal laser scan microscope (Leica Microsystems, Wetzlar, Germany) and an HC PL APO CS2 63x/1.4 oil objective. For quantitative analysis, maximum intensity projections were generated from all Z-stacks, which were captured for each condition with identical exposure times or laser settings.