Labtec 2 chamber slides

Cells grown on coverslip or in labtec II chamber slides (Nunc) were fixed in 4% paraformaldehyde for 1 h at room temperature, washed with PBS, and permeabilized in 0.1% Triton X-100 for 5 min or 0.5% saponin for 5 min. After incubation in blocking solution (5% BSA, 1% fish skin gelatin, 50 mM Tris in PBS) for 1 h, cells were incubated with primary antibodies in blocking solution for 1 h, washed and incubated with secondary antibodies for 1 h. When cells were permeabilized with saponin we added 0.1% saponin to the antibody staining solutions. For lipid droplet staining, fixed cells were stained for 20 min with HCS LipidTox Red neutral lipid stain (Invitrogen), diluted 1000-fold in PBS solution. Coverslips were embedded in Mowiol (Calbiochem) mounting medium [41] (link).

For IHC deparaffinized formalin fixed and paraffin embedded PCa tissue and fixed PCa cell lines grown on Lab-TECII chamber slides (154526, Nalge Nunc international, Naperville IL 60563–1796, USA) were used. Duplicate PCa tissue slides were obtained from the pathology department of patients with confirmed PCa as defined by a trained pathologist. The protocol used for IHC was previously described in [21 (link)]. It only diverted for the deparaffination step. In short, to damask the cells and tissue they were heated with TEG buffer (Tris 6,06g, EGTA 0,959 in 5L, pH 8,5) in a pressure cocker (Biocare medical decloaking chamber, Concord, Ca, USA). The slides were then blocked with 1% (v/v) H₂O₂ in MeOH for 30 minutes and with 5% BSA (w/v) in horse serum (20% v/v) (ImmPRESS, Vector Laboratories, Burlingame, CA) and Tris buffered saline (80% v/v) before application of antibody. The LHCGR OASG04237 antibody (ProMab biotechnologies Inc. Catalog # 30751) was diluted 1:7500 for the tissue and 1:200 for the cell lines and left overnight at 4°C followed by 1 h at room temperature. Anti-mouse secondary antibody (ImmPRESS, MP-7402) was applied for 30 minutes at room temperature and development was achieved with AEC (3-amino-9-cabazole, ImmPRESS, SK4205). Omission of primary antibody was used as control on sections of prostate, testis tissue and the cell lines.