Histochoice

Tissue biopsies were fixed overnight in HistoChoice^® (Amresco) and embedded in paraffin. Five micrometer-thick sections were stained with Masson's trichrome using Weigert's hematoxylin, fuchsin-ponceau, and aniline blue.
For immunodetection, biopsies were embedded in Tissue-Tek OCT Compound (Sakura Finetek) and frozen in liquid nitrogen. Immunofluorescence assays were performed on 5 μm-thick cryosections fixed with acetone (10 min at −20°C) as previously described.^{14 (link)} Cell nuclei were counterstained with Hoechst reagent 33258 (Sigma-Aldrich). The antibodies used were as follows: mouse monoclonal anti-K10 clone RKSE60 (Cedarlane), anti-human filaggrin (Abcam), anti-human K19 clone A53-B/A227 (gift from U. Karsten, Institute of Biological Sciences, University of Rostock, Germany), anti-integrin alpha-3 (VM2) clone HB-8530 (ATCC), anti-laminin-5 (alpha-3 subunit) conjugated to fluorescein isothiocyanate (FITC) (gift from P. Rouselle, IBCP, Lyon), anti-human Ki67 (Pharmingen), anti human leukocyte antigen A, B, C (HLA-ABC), conjugated to FITC (EMD Millipore), and rabbit anti-human type IV collagen (gift from J.A. Grimaud, Pasteur Institute, Lyon, France).

Slides were fixed with Histochoice (Amresco). The DeadEnd^™ Fluorometric TUNEL System (Promega, Madison, WI) was used for TUNEL assay according to the manufacturer’s instruction. Nuclei of cells were counterstained with DAPI (Vector Labs). For MVD assay, slides were labeled overnight with primary antibodies. Fluorescent IHC was performed first using monoclonal mouse antihuman anti-CD31 antibody (1:200, Dako), anti-EGFR antibody (1:250, Abcam), anti-BrdU antibody (1:250, Abcam), anti-Ki67 antibody (1:200, Abcam) or isotype control antibodies followed by FITC conjugated goat-anti-mouse IgG (1:1000, Abcam) or Cy5 conjugated goat-anti-mouse IgG (1:1000, Abcam) according to manufacturer’s protocols. The images were analyzed on the BioView Duet fluorescent scanning station using oil objective and DAPI/FITC/Rhodamine single band filters (Semrock). For each piece of the slide, the experiment was performed for three independently times.

HFF cells were cultured on circular micro cover glass (Electron Microscopy Sciences) and were infected with parasites for 18–30 h. For localization of rhoptry proteins to the rhoptry organelles, infected cells were fixed with Histochoice (Amresco), and permeabilized in 0.1% saponin (Sigma) for 10 min. For localization of rhoptry proteins to the PVM, infected cells were fixed in 4% paraformaldehyde and exposed to 0.002% digitonin for 5 minutes as previously described [62 (link)] to expose proteins associated with the cytosolic face of the PVM. All samples were blocked with 10% FBS and incubated with a 1:500 dilution of primary rabbit monoclonal α-HA-tag antibodies (Cell Signaling) 1 h at room temperature. Preparations were washed 3 times with PBS and incubated 1 h at RT with a 1:1000 dilution of secondary goat anti-rabbit IgG antibodies conjugated to Alexa Fluor 488. Samples were mounted in Slowfade Gold antifade with DAPI (Life Technologies) and then imaged with a Nikon A1R SI confocal microscope (Nikon, Inc.). Confocal images were processed with FIJI [129 (link)]. Vacuole locations were determined by differential interference contrast (DIC) microscopy.

Expression of D₄ receptors in thoracic aorta from SD rats was examined by laser scanning confocal microscope. Briefly, the aorta was cleared of blood with ice-cold oxygenated saline and kept in Histochoice (Amresco, Solon, OH) for one to two days at 4°C, then sectioned (4 μm), embedded in paraffin, and mounted on slides. The sections were double-immunostained with mouse anti-D₄ receptor antibody (1:100) and rabbit polyclonal anti-α-SM-actin antibody (1:100). The colocalization of D₄ receptors and α-SM-actin was performed as described previously [18 (link)].

Transfected cells were grown overnight after seeding on to glass coverslips, treated as described, and fixed 15-min at room temperature with Histochoice (Amresco). Coverslips were washed with PBS supplemented with 0.05% Tween. Blocking and antibody dilutions were carried out in PBS supplemented with 0.05% Tween and 2% BSA. Cx32 was probed with mouse anti-Cx32 clone 7C6.6 (1:1200). Signal was detected with Alexa-Fluor-conjugated secondary antibodies (Molecular Probes) and the coverslips were mounted using ProLong Gold plus DAPI (Molecular Probes) or Fluoromount (Invitrogen) with DAPI.
For proliferation studies, cultures were treated with BrdU (10 μM, Molecular Probes) overnight and fixed with Histochoice as described. Immunofluorescent detection of Cx32 preceded denaturation and immunolabeling for BrdU. Following immunolabeling for Cx32, samples were denatured 30 min with 2 N HCl in 0.1% Triton-X-100, neutralized with 0.2 M Borate buffer, pH 9.0 and washed with PBS. The samples were then processed for BrdU incorporation using monoclonal anti-BrdU (LabVision/ThermoFisher;1:150) immunofluorescence.