Non-Specific Binding, a Limitation of the Immunofluorescence Method to Study Macrophages In Situ

Abstract

Advances in understanding tissue regenerative mechanisms require the characterization of in vivo macrophages as those play a fundamental role in this process. This characterization can be approached using the immuno-fluorescence method with widely studied and used pan-markers such as CD206 protein. This work investigated CD206 expression in an irradiated-muscle pig model using three different antibodies. Surprisingly, the expression pattern during immunodetection differed depending on the antibody origin and could give some false results. False results are rarely described in the literature, but this information is essential for scientists who need to characterize macrophages. In this context, we showed that in situ hybridization coupled with hybridization-chain-reaction detection (HCR) is an excellent alternative method to detect macrophages in situ.

Keywords: hybridization-chain-reaction; in situ hybridization; macrophages.

Figure 1

Detection of CD206 marker expression in cells in IR-ASC muscle by immunofluorescence method. (A) Anti-CD206 (1) (Alexa488, turquoise fluorescence), (B) Anti-CD206 (2) (Alexa488, red fluorescence), (C) Anti-CD206 (3) (Alexa488, yellow fluorescence) labeling, (D) Nuclear staining with DAPI (blue fluorescence). (E) Merged image, (F–H) expanded view: high magnification image of the area within the red rectangles in image E. (I) Venn diagram of CD206-positive cells of 3000 cells. Thin arrow: cells labeled with three antibodies; thick arrow: cells labeled with Anti-CD206 (2) antibody only; arrowhead: cells labeled with Anti-CD206 (3) antibody only.

Figure 2

Negative controls of the CD206 marker expression in cells in IR-ASC muscle by immuno-fluorescence method. (A) Negative control of Figure 1A with Anti-CD206 (1) (Alexa488, turquoise fluorescence). (B) Negative control of Figure 1B with Anti-CD206 (2) labeling (Alexa488, red fluorescence). (C) Negative control of Figure 1C with Anti-CD206 (3) labeling (Alexa488, yellow fluorescence) labeling. (D) Negative control of Figure 1D with nuclear staining with DAPI (blue fluorescence). (E) Merged image.

Figure 3

In situ fluorescence microspectroscopy analysis of the Anti-CD206 (1, 2, 3) antibodies in IR-ASC muscle. Emission spectra of Anti-CD206 (1)/Alexa Fluor 488 (turquoise line), Anti-CD206 (2)/Alexa Fluor 568 (red line) and Anti-CD206 (3)/Alexa Fluor 647 (yellow line).

Figure 4

Detection of CD206 expression in the IR-ASC muscle by in situ hybridization. (A–F) In situ-DIG; (G,H) In situ-HCR. (A) Negative control. (B) Expression of β-actin mRNA (positive control). (C) Expression of CD206 mRNA. (D–F) For the quantification, slide was post-stained with DAPI. (D) Expression of CD206 mRNA. (E) Nuclear staining with DAPI (blue fluorescence). (F) Both CD206 mRNA and DAPI staining. (G) Expression of CD206 mRNA. (H) Negative control of G. Probe-CD206 (Alexa546, red fluorescence), nuclear staining with DAPI (blue fluorescence).