EFB Dengue Literature Review

Immunohistochemistry

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Immunohistochemistry

Overview

Immunohistochemistry (IHC) is a tissue-based technique that uses antibody–antigen interactions to visualise the spatial distribution of specific proteins within tissue sections. In the context of B cell biology, IHC is essential for determining the anatomical localisation of B cell subsets — distinguishing between germinal centre, follicular, and extrafollicular (T zone–red pulp border) compartments. IHC provides the spatial resolution that flow cytometry and BCR sequencing lack: it can demonstrate that B cells are physically present at a particular anatomical site, interacting with specific cell types (T cells, dendritic cells, FDCs), and undergoing proliferation in situ.

Key Points from Literature

  • Core method for demonstrating extrafollicular B cell localisation: William2002 used multi-colour IHC on spleen sections from MRL/lpr mice to show that RF (rheumatoid factor) B cells localise to the T zone–red pulp border rather than GCs. Markers used: anti-idiotype 4-44 (RF B cells), CD3 (T cells), PNA (GCs), CR1 (FDCs), CD11c (dendritic cells), BrdU (proliferation), Thy1.2 (T cells), DAPI (nuclei). Adjacent serial sections enabled co-localisation analysis between different marker combinations (see William2002 - Extrafollicular Somatic Hypermutation in Autoimmune Mice, multi-colour IHC on frozen spleen sections, 8 mice).
  • IHC resolves ambiguities that flow cytometry cannot: Flow cytometry can identify expanded EF-phenotype cells (e.g., CD21⁻CD11c⁺ B cells in dengue per Ansari2025) but cannot determine their anatomical location within lymphoid tissue. William2002’s IHC was essential for proving that mutating B cells were physically at the T zone–red pulp border, not in residual GCs — a distinction that defined the field (see William2002 - Extrafollicular Somatic Hypermutation in Autoimmune Mice).
  • BrdU pulse-labelling quantifies in situ proliferation: A 2-hour BrdU pulse followed by IHC co-staining with anti-Id demonstrated that 15% of RF Id⁺ B cells at the EF site were actively proliferating — establishing the kinetics of the EF response directly in tissue (see William2002 - Extrafollicular Somatic Hypermutation in Autoimmune Mice).

Contradictions & Debates

None documented in current wiki sources.

Extrafollicular Response, Germinal Center, CD11c, BCR Sequencing, FACS Sorting

Sources

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