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. 2000 Jul;53(7):518–524. doi: 10.1136/jcp.53.7.518

Immunohistowax processing, a new fixation and embedding method for light microscopy, which preserves antigen immunoreactivity and morphological structures: visualisation of dendritic cells in peripheral organs

B Pajak 1, T De Smedt 1, V Moulin 1, C De Trez 1, R Maldonado-Lopez 1, G Vansanten 1, E Briend 1, J Urbain 1, O Leo 1, M Moser 1
PMCID: PMC1731227  PMID: 10961175

Abstract

Aims—To describe a new fixation and embedding method for tissue samples, immunohistowax processing, which preserves both morphology and antigen immunoreactivity, and to use this technique to investigate the role of dendritic cells in the immune response in peripheral tissues.

Methods—This technique was used to stain a population of specialised antigen presenting cells (dendritic cells) that have the unique capacity to sensitise naive T cells, and therefore to induce primary immune responses. The numbers of dendritic cells in peripheral organs of mice either untreated or injected with live Escherichia coli were compared.

Results—Numbers of dendritic cells were greatly decreased in heart, kidney, and intestine after the inoculation of bacteria. The numbers of dendritic cells in the lung did not seem to be affected by the injection of E coli. However, staining of lung sections revealed that some monocyte like cells acquired morphological and phenotypic features of dendritic cells, and migrated into blood vessels.

Conclusions—These observations suggest that the injection of bacteria induces the activation of dendritic cells in peripheral organs, where they play the role of sentinels, and/or their movement into lymphoid organs, where T cell priming is likely to occur.

Key Words: dendritic cell • Escherichia coli • immunohistochemistry

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Figure 1 Immunostaining of major histocompatibility complex (MHC) class II positive or CD11c positive cells in organs of mice injected or not injected with bacteria. Heart (A and B), intestine (C and D), kidney (E and F), and lung (G and H). Sections from Balb/c mice injected 24 hours previously with phosphate buffered saline (A, C, E, and G) or Escherichia coli (B, D, F, and H) were stained with anti-I-Ed (A–F) or anti-CD11c (G and H) monoclonal antibodies, and further counterstained with haemalun. Scale bar, 50 µm (A, B, C, D, G, and H), 100 µm (E and F).

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Figure 2 Histopathology of the lung after Escherichia coli injection. Lungs from Balb/c mice injected with phosphate buffered saline (A and C) or E coli (B, D, and E) one hour previously were fixed, dehydrated, and embedded according to the Immunohistowax processing protocol and stained with haemalun-eosin. Note the infiltration of neutrophils in alveoli (D) and in arterioles (E). Scale bars, 250 µm (A and B); 25 µm (C–E).

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Figure 3 Phenotype and morphology of lung dendritic cells after the inoculation of bacteria. Immunohistowax processed lung sections from mice injected with phosphate buffered saline (A) or live Escherichia coli (B–E) were double stained with anti-CD11c in red and anti-I-Ed in blue. Note the CD11c positive, major histocompatibility complex class II positive cells in purple (B–E). Scale bars, 100 µm (A and B); 50 µm (C–E).

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