Mouse igg1

Three μm formalin-fixed, paraffin-embedded sections were immunostained for TTF1 (Abcam, clone 2Cla, 1:50, pretreated with citrate buffer for 20 min at 100°C), p63 (Leica-Novocastra, clone 7 Jul, 1:40, pretreated with bond 2 for 30 min at 100°C), napsin (Leica-Novocastra, clone IP64, 1:400, pretreated with bond 2 for 30 min at 95°C), p40 (Biocare Medical, polyclonal antibody, 1:100) and TP53 (Dako, clone DO-7, 1:200, pretreated with bond 2 for 20 min at 95°C) using a Bond Max autostainer (LEICA Bond III platform) from Leica Microsystems (Wetzlar, Germany) and counterstained with haematoxylin. Mouse IgG1 (Dako, 1:20) was used as a negative control.

Purity of the eSC suspensions was characterized by immunostaining for stromal and epithelial cell markers using the UltraVision One Detection System HRP Polymer kit (Fisher Scientific TL-060-HLJ) for a chromogenic reaction. The primary antibody anti-vimentin clone V9 (DAKO, Agilent, USA) at 1:250 dilution was used to stain stromal cells, while the primary antibody anti-cytokeratin-8/18 clone Zym5.2 (Thermofisher, USA) at 1:100 dilution was used to stain epithelial cells. Mouse IgG1 (Dako, Agilent, USA) at 1:100 dilution served as a negative control. 100 000 cells/well were plated in an 8-well chamber slide and incubated overnight. The cells were fixed with 4% buffered formaldehyde solution and permeabilized with cold ethanol at -20°C. Hydrogen peroxide block was performed with 0.3% H₂O₂ in methanol. Immunostaining was done according to manufacturer’s protocol and the results were recorded with light microscopy.

Biopsies were embedded in OCT Tissue TEK and stored in liquid nitrogen. Frozen sections were cut (5 μm) using a cryostat, after which sections were stored at −80 °C until further use. For staining, sections were thawed and air-dried at room temperature and subsequently fixed with acetone [34 (link)]. Sections were washed and stained with monoclonal primary antibodies or isotype controls: rat anti-human CXCR5-Alexa Fluor 488 IgG2b (BD Biosciences), rabbit anti-human Bcl-6 IgG (Bio SB, Santa Barbara, CA), mouse anti-human ICOS IgG1 (Novo Biologicals, Littleton, CO, USA), rat IgG2b Alexa Fluor 488 (isotype control, BD Biosciences), mouse IgG1 (isotype control, Dako Cytomation, Heverlee, Belgium), or rabbit IgG (isotype control, Dako Cytomation) diluted in PBS/1% BSA/10% normal human serum (NHS; Lonza, Basel, Switzerland) overnight at 4 °C. After washing with PBS, (directly labeled) secondary antibodies (goat anti-rabbit IgG Alexa 546 and goat anti-mouse IgG1 Alexa 633, both from Life Technologies, Bleiswijk, The Netherlands) were incubated for 30 min in PBS/1%BSA/10% NHS. After washing with PBS, slides were covered with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA) and analyzed on a confocal imaging microscope (Leica Microsystems, Wetzlar, Germany).

Antibodies for ß-Actin, von Willebrand factor (vWF), GAPDH, and PDZK1 were purchased from Abcam (Cambridge, UK). Goat-anti-rabbit and goat-anti-mouse negative control antibodies were obtained from Biorad (Hercules, CA, USA). SR-BI was detected using a sequence specific SR-BI antibody purchased from Abcam (Cambridge, UK). For double-fluorescence immunohistochemistry monoclonal mouse-anti-human Desmin, mouse IgG1, and normal rabbit immunoglobulin fraction were obtained from Dako (Glostrup, Denmark). Goat-anti-mouse CyTM3 and donkey-anti-rabbit CyTM2 were purchased from Jackson Immunoresearch, Dianova (Hamburg, Germany). The polyclonal rabbit-anti-human SR-BI was obtained from Abcam (Cambridge, UK). Primers for SR-BI and the ribosomal protein L30 gene were designed using Primer3 software (http://frodo.wi.mit.edu/ (accessed on 19 October 2012; newer version available https://bioinfo.ut.ee/primer3/)) and were purchased from Ingenetix (Vienna, Austria). All primers were chosen to span exons to avoid the amplification of traces of contaminating genomic DNA.