Cyclin d1

Immunofluorescence stainings were performed as described [22 (link), 32 (link)]. For PML immunostaining, cells were fixed with 4% formaldehyde for 10 min and permeabilized with 0.25% Triton X-100 for 5 min. Antibodies against PML (Milipore, MA), PCNA (Santa Cruz Biotechnology, Santa Cruz, CA) and cyclin D1 (Nichirei Bioscience, Tokyo, Japan) and secondary antibodies conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR) or Cy-3 (Jackson ImmunoResearch Laboratories, West Grove, PA) were used. Cells were counterstained for DNA with Hoechst 33258 (4 μg/mL in Vectashield mounting medium; Vector Laboratories, Burlingame, CA). Images were captured using a CCD camera attached to a fluorescence microscope (Keyence, Osaka, Japan). For each data point, > 50 cells were counted from at least three independent samples.

Serial sections (4 μm) were cut from the block of paraffin-embedded tissue, stained with hematoxylin and eosin, and used for the following immunohistochemical stains: CD20 (L26 [1:400]; DAKO, Glostrup, Denmark), CD3 (LN10 [1:200]; Novocastra, Newcastle, UK), CD5 (4c7 [1:50]; Novocastra), CD10 (56C6 [100:1]; Novocastra), CyclinD1 (SP4 [1:50]; Nichirei, Tokyo, Japan), Ki-67 (MIB-1 [1:2500]; Novocastra), IgG (polyclonal [1:20,000]; DAKO), and IgG4 (HP6025 [1:10000]; The Binding Site, Birmingham, UK). Following immunostaining using an automated Bond Max stainer (Leica Biosystems, Melbourne, Germany), the numbers of IgG4⁺ and IgG⁺ cells were estimated in areas with the highest density of IgG4⁺ cells. In accordance with the consensus statement on the pathological features of IgG4-RD17 (link), three different high-power fields (HPFs) (total magnification, ×400) were examined to calculate the average number of IgG4⁺ cells per HPF and the IgG4⁺/IgG⁺ cell ratio. In situ hybridization was also performed for κ and λ-light chains (Leica Biosystems) using a Bond Max stainer.