Immunocytochemistry and immunofluorescence detection procedures
Single label immunocytochemistry was performed on paraffin embedded sections (5 μm) and specific antibody binding was detected with biotinylated secondary antibodies and streptavidin-peroxidase conjugates (ABC System; Vector Diagnostics, Burlingame, CA), as described previously [10, 21]. Tissues were counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO) and mounted in Permount (Fisher Scientific, Fair Lawn, NJ). The specificity of staining for individual antibodies was confirmed using either an unrelated isotype control antibody or secondary antibody alone. For some antibodies, where the target epitope was known to be destroyed by paraffin embedding, expression profiles were determined by staining frozen tonsil sections that were fixed in STF immediately prior to use.
Double label detection of target antigens was performed by immunofluorescence staining of paraffin embedded or frozen tissue sections, taking into account the properties of the primary antibodies. In short, after antigen retrieval by citrate buffer, tissue sections were blocked with TNB [0.1 M Tris. HCl pH7.5, 0.15 M NaCl, 0.5% w/v Dupont blocking reagent (Perkin Elmer, Boston, MA)] and 1.5% horse serum followed by overnight incubation with primary antibody at 4°C. Tissue sections were then washed, reblocked and incubated with appropriate secondary antibody (Alexa 568-anti mouse or Alexa 488-anti rabbit conjugates; Molecular Probes Inc, Eugene, OR) for 1 h at room temperature. After dehydration through graded alcohols, the sections were cleared with methyl salicylate (Sigma-Aldrich), mounted in DEPEX (Electron Microscopic Sciences, Ft. Washington, PA) and stored at 4°C until viewed. Fluorescent images were collected with a Zeiss upright microscope equipped with a Spot Camera and motorized stage for high resolution capture of brightfield and fluorescence images and then processed with Adobe Photoshop (Abode Systems Inc, San Jose, CA).
The following antibodies were used at the dilutions indicated: CXCR4 (1:100, clone12G5; BD Pharmingen, San Diego, CA); CXCR4 (1:100, rabbit polyclonal; eBioscience, San Diego, CA), CCR5 (1:750, clone 45549.111; R&D Systems, Minneapolis, MN); Heparan sulfate (1:250, clone F58-10E4; Seikagaku Corp., Tokyo, Japan); CD3 (1:500, rabbit polyclonal; DAKO, Carpenteria, CA); CD4 (1:50, clone IF6; Zymed, San Francisco, CA); CD45RO (prediluted sample, clone UCHL-1; BioGenex, San Ramon, CA); cytokeratin (rabbit polyclonal DAKO); CD54/ICAM-1 (1:50, clone LB-2; BD Pharmingen); CD11a/LFA-1 (1:50, clone G43-25B; BD Pharmingen); DC SIGN (1:50, clone DCN46; BD Pharmingen); S100 (1:5000, rabbit polyclonal antibody; DAKO); IL-8 (1:250, rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA); Hsp27 (1:250, clone G3.1; BioGenex); GalCer (1:100, clone MAB342; Chemicon, Temecula, CA). The optimal dilution for each antibody was determined empirically in preliminary titration experiments.