Immunohistochemical Analysis of Fixed Tissues

In situ hybridization of formalin-fixed tissues and immunohistochemistry of paraformaldehyde-fixed tissues were performed as described [32 (link)]. The primary antibodies used for immunohistochemistry were against calprotectin (MAC 387; ThermoFisher Scientific), CD163 (GHI/61; Invitrogen), cytokeratin (AE1/AE3; Abcam), cleaved GSDMD (EPR20829-408; Abcam), histone H3 (ab18521; Abcam), IFN-1α (MMHA-2; Fisher), IL-1β (6E10; Novus Biologicals), MxA/Mx1 (4812; Novus Biologicals) and influenza A nucleoprotein (DPJY03, BEI Resources). To quantify expression in tissue, TIFF images taken by confocal microscopy were analyzed using Nikon NIS Elements version 4.50.00. Regions of interest were drawn to encapsulate lung tissue and exclude edges of tissue/airways in the tissue where no tissue is present. Microscope slides were imaged non-repetitively for 10 images per tissue section. Stains of interest were then quantified by recording the binary area (recorded as pixels) and averaging the values to generate individual data points per animal, which were averaged for presentation as single data points.

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Corry J., Kettenburg G., Upadhyay A.A., Wallace M., Marti M.M., Wonderlich E.R., Bissel S.J., Goss K., Sturgeon T.J., Watkins S.C., Reed D.S., Bosinger S.E, & Barratt-Boyes S.M. (2022). Infiltration of inflammatory macrophages and neutrophils and widespread pyroptosis in lung drive influenza lethality in nonhuman primates. PLoS Pathogens, 18(3), e1010395.

Publication 2022

MAC 387 AE1/AE3; EPR20829-408 ab18521

Animal Antibodies Biologicals Calprotectin Cd163 Confocal microscopy Cytokeratin Formalin Histone h3 Il 1β Immunohistochemistry In situ hybridization Influenza Lung Microscope Novus Nucleoprotein Paraformaldehyde Stains Tissue

Corresponding Organization : Emory University

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Variable analysis

independent variables

Tissue fixation type (formalin-fixed or paraformaldehyde-fixed)

dependent variables

Expression of calprotectin (MAC 387), CD163, cytokeratin, cleaved GSDMD, histone H3, IFN-1α, IL-1β, MxA/Mx1, and influenza A nucleoprotein in lung tissue

control variables

Microscope settings (confocal microscopy)
Number of images per tissue section (10 images)
Regions of interest (lung tissue excluding edges and airways)

positive controls

None specified

negative controls

None specified

Annotations

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