Comprehensive Immunohistochemical Analysis of Lymph Nodes

Immunohistochemistry was performed as previously indicated [73 (link), 82 (link)] on formalin-fixed paraffin-embedded (FFPE) LNs obtained at necropsy. Briefly, sections were deparaffinized and antigen retrieval was performed using a Retriever (Electron Microscopy Services, Hatfield, PA) in Tris-EDTA-Tween-80 buffer 73. Sections were stained for Tcells/B cells/dendritic cells (polyclonal rabbit anti-CD3, Dako, Santa Clara, CA; polyclonal rabbit anti-CD20, Thermo Fisher Scientific, Pittsburgh, PA; mouse-anti-CD11c, Leica Microsystems, Buffalo Grove, IL), macrophage subsets (mouse anti-CD68, Thermo Fisher; rabbit anti-DC-SIGN, ProSci Inc, Poway, CA; mouse anti-CD163, Thermo Fisher), LN vascular and structural aspects (Goat anti-LYVE-1, R&D Systems, Minneapolis, MN; rat-anti PNAd, BioLegend, San Diego, CA), and LN conduit systems (visualized by staining for rabbit anti-collagen 1 [Abcam, Cambridge, MA]). Primary antibodies were visualized with species- and isotype-specific secondary antibodies purchased from Jackson ImmunoResearch (West Grove, PA). Auramine rhodamine was performed as previously indicated [73 (link)] using reagents from BD Biosciences (San Jose, CA). Images were acquired at 20x magnification with a Nikon e1000 widefield microscope (Nikon, Melville, NY) with Nikon Elements.

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Ganchua S.K., Cadena A.M., Maiello P., Gideon H.P., Myers A.J., Junecko B.F., Klein E.C., Lin P.L., Mattila J.T, & Flynn J.L. (2018). Lymph nodes are sites of prolonged bacterial persistence during Mycobacterium tuberculosis infection in macaques. PLoS Pathogens, 14(11), e1007337.

Publication 2018

Retriever polyclonal rabbit anti-CD3 mouse anti-CD68 mouse anti-CD163 Goat anti-LYVE-1 rat-anti PNAd rabbit anti-collagen 1

Anti cd3 Antibodies Antigen Auramine B cells Buffalo Cd163 Collagen 1 Dc sign Dendritic cells Edta Electron microscopy Formalin Goat Immunohistochemistry Isotype Macrophage Microscope Mouse Necropsy Paraffin Pnad Rabbit Rhodamine Tris buffer Tween 80 Vascular

Corresponding Organization : University of Pittsburgh

Other organizations : Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center

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Variable analysis

independent variables

Immunohistochemistry staining protocols
Tissue samples (formalin-fixed paraffin-embedded (FFPE) LNs obtained at necropsy)

dependent variables

Presence and localization of T cells, B cells, dendritic cells, macrophage subsets, lymph node (LN) vascular and structural aspects, and LN conduit systems

control variables

Tris-EDTA-Tween-80 buffer for antigen retrieval
Species- and isotype-specific secondary antibodies for visualization
Auramine rhodamine staining as previously indicated

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