Immunohistochemistry was conducted using the avidin-biotin technique as described previously17 (link), 18 (link). The primary antibodies were polyclonal rat anti-macrophages/ monocytes (MOMA-2, Abcam, ab33451, 1:200), polyclonal rabbit anti-inducible nitric oxide synthase (iNOS, Abcam, ab15323, 1:200), polyclonal rabbit anti-YM-1 (Abcam, ab93034, 1:200), monoclonal rabbit anti-ferritin (Abcam, ab75973, 1:200), and polyclonal rabbit anti-heme oxygenase-1 (HO-1, Enzo, SPA-895-F, 1:500). Negative controls omitted the primary antibody. In this study, we also use hematoxylin counterstaining for the nucleus detection. Briefly, the sections used for immunohistochemistry was immersed into hematoxylin for 10 seconds right after the 3,3’-diaminobenzidine (DAB) incubation. An oil immersion lens was adopted for morphological observations.
Protocol detail
Immunohistochemical Analysis of Macrophage Markers
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Antibodies
Antibody
Avidin
Biotin
Ferritin
Hematoxylin
Heme oxygenase 1
Immersion
Immunohistochemistry
Inducible nitric oxide synthase
Inos
Lens
Macrophages
Monocytes
Nucleus
Rabbit
Seconds
Corresponding Organization : University of Michigan–Ann Arbor
Other organizations : Air Force Medical University, Xijing Hospital
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Variable analysis
independent variables
- Primary antibodies used: polyclonal rat anti-macrophages/ monocytes (MOMA-2), polyclonal rabbit anti-inducible nitric oxide synthase (iNOS), polyclonal rabbit anti-YM-1, monoclonal rabbit anti-ferritin, and polyclonal rabbit anti-heme oxygenase-1 (HO-1)
- Expression/localization of macrophages/monocytes, inducible nitric oxide synthase, YM-1, ferritin, and heme oxygenase-1 as detected by immunohistochemistry
- Avidin-biotin technique for immunohistochemistry as described previously
- Hematoxylin counterstaining for nucleus detection
- Oil immersion lens for morphological observations
- Negative controls omitted the primary antibody
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