Peroxidase conjugated streptavidin

Paraffin-embedded tumor sections and a series of control organs from the mice used for ¹²³I- scintigraphy were immunohistochemically stained as described previously 51 (link). Staining was performed using a primary mouse monoclonal NIS-specific antibody (Merck Millipore; dilution 1:500) for 90 min followed by a biotin-SP-conjugated goat anti-mouse IgG antibody (Jackson Immunoresearch; dilution 1:200) for 20 min and then peroxidase-conjugated streptavidin (Jackson Immunoresearch; dilution 1:300) for an additional 20 min. Immunohistochemically stained tumor sections were scanned using the Pannoramic MIDI digital slide scanner and pictures taken with Caseviewer software (3DHISTECH Ltd., Budapest, Hungary) and control organs were imaged on an Olympus BX41 microscope equipped with an Olympus XC30 CCD camera (Olympus, Shimjukum Tokio, Japan).

Gold particles of 5 nm coupled to bovine serum albumin (BSA-Au^{5 (link)}), and 10 nm or 15 nm gold particles conjugated to protein A were made on site (Cell Microscopy Core, UMC Utrecht, The Netherlands). HA-Vps8, Vps8-V5 and Vps8-GFP, GFP-Rab4, mCherry-Rab4, FLAG-Rab4, mCherry-Rab5, mCherry-Rab7, mCherry-Rab11 and untagged Vps33B were cloned from cDNA purchased from Origen. HA-Vps3, GFP-Vps3, GFP-Vps11 and Vps41-GFP were a generous gift from Dr. J. Neefjes (NKI, Amsterdam, The Netherlands), VIPAS39-mCherry was a generous gift from Dr. P. Gissen (LMCB, UCL, London), and Vps33B-HA-V5-His was a generous gift from Dr. V. Faundez (Cell Biology, Emory University, Atlanta, GA, USA). Vps8, Vps3, Vps33B and VIPAS39 SMARTPool siRNAs were purchased from Dharmacon. FN and type-I collagen were purchased from Sigma. MG132 was purchased from Enzo Life Sciences. EZ-link Sulfo-NHS-SS-biotin was from Thermo Fisher. Complete protease inhibitors were purchased from Roche. For detection of in vitro-translated proteins, peroxidase-conjugated streptavidin (0.1 µg/ml) was used (Jackson ImmunoResearch).

Boiled cell lysates (10 μl) were loaded onto a NuPAGE 4–12% BisTris SDS-polyacrylamide gel (Invitrogen) and then transferred to polyvinylidene fluoride membranes (VWR) for 1 h at 100 mV. Blots were blocked using 5% bovine serum albumin (US Biologicals) in 50 mm Tris, pH 8.0, containing 150 mm NaCl and 0.5% Tween buffer (TBST) for 2 h at 4 °C. Biotinylated anti-maltose-binding protein antibody (Vector Laboratories) was diluted 1:1500 in 1% bovine serum albumin in TBST and incubated overnight at 4 °C. Blots were washed with TBST for 1 h with three buffer exchanges at room temperature before incubation with secondary antibody in 1% bovine serum albumin in TBST at 1:1500 dilution, peroxidase-conjugated streptavidin (Jackson ImmunoResearch) for 2 h at room temperature. Blots were again washed for 1 h using three exchanges of TBST. A 1:1 mixture of buffers from the Pierce ECL Western blotting substrate kit was added to the blot, and chemiluminescence was measured using an ImageQuant LAS 4000 (GE Healthcare).

Endogen peroxidases were blocked with 0.5% H₂O₂ in PBS for 10 min at room temperature. After repeated washes in PBS, the endogen biotin was blocked with ready-to-use reagents (Biotin-Blocking System, Dako, Carpinteria, CA, USA): samples were incubated for 10’ with avidin 0.1% solution, washed in distilled water, and incubated for 10’ with biotin 0.01% solution. After repeated washing in distilled water, samples were then pre-incubated with a blocking buffer (0.2% bovine serum albumin, BSA, and 0.2% Triton-X, in PBS) for 60 min at room temperature. The cells were then incubated in hyaluronic acid binding protein (HABP), Bovine Nasal Cartilage, Biotinylated, (Merck Life Science S.r.l., Milano, Italy), diluted 1:1000 in the same pre-incubation buffer, and maintained overnight at 4 °C. After repeated PBS washing, cells were incubated for 30’ in peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc.), diluted 1:250 in the same pre-incubation buffer. The reaction was then developed with 3,3′- diaminobenzidine (Liquid DAB plus substrate Chromogen System kit; Dako) and stopped with distilled water. Negative controls were carried out by omitting- incubation with HABP, confirming the specificity of the immunostaining. Nuclei were counterstained with hematoxylin, ready to use (Dako).