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Fluorescence Compared to Brightfield Detection of CD35 in Lymphoid Follicles of the Monkey to Assess Immunomodulatory Treatment Effect
Author(s) -
Brodbeck Jens,
Chu Felix,
Edick Melissa Gonzales,
Rangell Linda,
Bheddah Sheila,
Cain Gary
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.655.10
Subject(s) - germinal center , follicular dendritic cells , biology , pathology , mannose receptor , fluorescence , polyclonal antibodies , antibody , immunology , microbiology and biotechnology , medicine , b cell , t cell , antigen presenting cell , biochemistry , immune system , macrophage , in vitro , physics , quantum mechanics
Aims Lymphoid follicles of secondary lymphoid organs are dynamic structures inducible by immunization and/or inflammatory stimulation. Follicular dendritic cells (FDC) are specialized antigen‐presenting cells exclusive to the germinal centers (GC) of lymphoid follicles. Within the GC, FDC's form a reticular network that sustains the architecture of lymphoid follicles and interact with the GC B cells via several mechanisms including complement receptors such as CD35. The expression of CD35 in lymphoid follicles is therefore a measure of the efficacy of immunomodulatory (IMM) treatment strategies. Methods CD35 in formalin‐fixed paraffin‐embedded spleen sections of cynologous monkeys was detected with a mouse monoclonal antibody and visualized using a polyclonal secondary antibody coupled to either horse‐radish peroxidase for DAB precipitation or the fluorophore Alexa647. Digital brightfield and fluorescence images (20x) were acquired on a Nanozoomer 2HT (Hamamatsu) and Ariol SL‐50 (Genetix) whole slide scanner respectively. Images were quantified using Definiens Developer software. Results Fluorescence significantly increased the dynamic range of CD35 compared to chromogen. Fluorescence signal intensities ranged from 0 – 256 compared to 0 – 0.8 using chromogen. Both detection methods showed a small but significant decrease in follicular CD35 following IMM treatment without resolving dose effects. However the increased dynamic range of fluorescent CD35 allowed a robust distinction between follicles containing germinal centers and those without. Thus a clear dose effect after IMM treatment could be resolved using fluorescence, but not chromogen for CD35 detection.