Figure 4. Immunohistochemical controls validating the specificity of CD11b and CD206 staining. Representative images of staining controls.

A) No primary antibody for CD206, B) Isotype control antibody for CD11b and C) No primary antibody for either CD11b or CD206. CD11b (green), CD206 (red) and cell nuclei/DAPI (blue). Scale bars = 100 μm. All images were acquired on serial sections from the same sample with a 20x objective using the same exposure settings. The same display adjustment was applied across all images. A. Images showing a lack of CD206 staining when the primary antibody for CD206 is not applied to the sections. This control shows the specificity of the CD206 antibody; staining is not a product of cross reactivity with reagents used to amplify CD11b or due to non-specific tissue staining. B. Lack of CD11b+ staining when an isotype control antibody is applied. Similar to panel A, this control shows the specificity of the CD11b antibody since the isotype-matched control does not produce non-specific staining. Additionally, this control shows that CD206 staining does not result from cross reactivity to CD11b reagents since no CD11b staining is present but CD206+ cells are clearly identified. C. Images show very low non-specific background staining of skeletal muscle tissue sections from the use of amplification reagents; thus positive macrophage staining can clearly be distinguished from tissue background.