Control mouse igg1

Mouse monoclonal antibodies against human CCTα, β, γ, δ, ε, ζ, η, θ, β-actin, AIB1, ubiquitin, and ERα, and goat anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The control mouse IgG1 was purchased from DAKO (Glostrup, Germany), and the CCT1-8 and ERα cDNA sequences were purchased from I.M.A.G.E. Consortium (Lawrence Livermore National Laboratory; Livermore, CA, USA). MG132 (the proteasome inhibitor) and all other reagents were purchased from Sigma (St. Louis, MO, USA).

Formalin-fixed and paraffin-embedded tissue sections were used. Antigen retrieval was performed as described above. The sections were incubated with a mixture of goat anti-human LAP (1:75 = 1.25 μg/mL) and mouse monoclonal anti-human CD83 (1:8; clone 1H4b, Novocastra-Leica Microsystems, Benton Lane, UK), anti-CD68 (1:80; clone PG-M1, DAKO) or anti-CD3 (1:8; clone F7.2.38, DAKO) overnight. Alexa Fluor 488-labeled donkey anti-goat IgG (1:100 = 20 μg/mL, Molecular Probe, Carlsbad, CA) and Alexa Fluor 555-labeled donkey anti-mouse IgG (1:100 = 20 μg/mL) were applied in a mixture for 30 min. After DAPI (Molecular Probe) nuclear staining, specimens were mounted with ProLong Gold (Molecular Probe). Immunofluorescent observation was performed with a confocal laser scanning microscope (TCS SP5, Leica Microsystems, Wetzlar, Germany) or with a Nikon E800 microscope (Nikon, Tokyo, Japan). For negative control, the primary antibodies were replaced by either non-immunized goat IgG (IBL; 1.25 μg/mL) or control mouse IgG1 (DAKO; 1:100 = 4 μg/mL).