Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies

Abstract

The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin-embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated.

Keywords: Biotinylated; Double stain; IHC; Ki-67; Macrophage; Rat.

Fig. 1

Schematic representation of protocol for staining formalin-fixed paraffin-embedded tissue by double immuno uorescence with two mouse monoclonal antibodies

Fig. 2

Double immunofluorescent staining of macrophages using two mouse monoclonal antibodies targeting CD68 and inducible nitric oxide synthase (iNOS) in formalin-fixed paraffin embedded colon from a colitic rat. Images depict serial sections inflammatory infiltrate within colonic mucosa damaged by colitis induction. Staining for iNOS (unconjugated primary antibody, green), CD68 (biotinylated primary antibody, red), and DAPI (blue) is shown in the first, second, and third columns, respectively. Overlays of the green, red, and blue channels are shown in the fourth column. a–d Staining for CD68, iNOS, and DAPI reveals the presence of several macrophages (CD68-positive cells) co-expressing iNOS. e–h The iNOS single-stain control (tissue did not receive CD68 antibody) reveals iNOS staining in areas of colocalization seen in a–d. i–l The CD68 single-stain control (tissue did not receive iNOS antibody) reveals CD68 staining in areas of colocalization as well as in areas negative for iNOS as seen in a–d. m–p Negative control tissue (did not receive either of the primary antibodies) demonstrates some autofluorescence that can be distinguished from areas of true positivity (orange color in p). Scale bar = 100µm

Fig. 3

Double immunofluorescent staining of macrophages using two mouse monoclonal antibodies targeting CD68 and inducible nitric oxide synthase (iNOS) in formalin-fixed paraffin embedded colon from a normal rat. Images depict serial sections of normal colonic mucosa and submucosa. Staining for iNOS (unconjugated primary antibody, green), CD68 (biotinylated primary antibody, red), and DAPI (blue) is shown in the first, second, and third columns, respectively. Overlays of the green, red, and blue channels are shown in the fourth column. a–d Staining for CD68, iNOS, and DAPI reveals the presence of several macrophages (CD68-positive cells) lacking iNOS staining. e–h The iNOS single-stain control (tissue did not receive CD68 antibody) confirms the lack of iNOS staining observed in a–d. i–l The CD68 single-stain control (tissue did not receive iNOS antibody) shows CD68 staining consistent with that shown in a–d. m–p Negative control tissue (did not receive either of the primary antibodies) demonstrates some autofluorescence that can be distinguished from areas of true positivity (orange color in p). Scale bar = 100µm

Fig. 4

Double immunofluorescent staining of macrophages using two mouse monoclonal antibodies targeting CD68 and Ki-67 in formalin-fixed paraffin embedded colon, liver, and brain from normal rats. Staining for Ki-67 (unconjugated primary antibody, green), CD68 (biotinylated primary antibody, red), and DAPI (blue) is shown in the first, second, and third columns, respectively. Overlays of the green, red, and blue channels are shown in the fourth column. a–d Staining for CD68, Ki-67, and DAPI in colonic tissue reveals the presence of several macrophages (CD68⁺ cells) in the lamina propria and abundant proliferating (Ki-67⁺) epithelial cells in the bottom half of the colonic crypts. e–h Staining for CD68, Ki-67, and DAPI in hepatic tissue shows various macrophages (CD68⁺ cells) in the interstitium and several proliferating (Ki-67⁺) hepatocytes, including mitotic figures. i–l Staining for CD68, Ki-67, and DAPI in the cerebral cortex demonstrates occasional CD68⁺ cells and Ki-67⁺ cells. m–p Staining for CD68, Ki-67, and DAPI in the CA2 region of the hippocampus demonstrates the presence of some proliferating cells (Ki-67⁺) and the occasional macrophage (CD68⁺). Scale bars = 50µm

Fig. 5

Artifactual co-localization resulting from interaction between tissue-bound anti-mouse secondary antibody and biotinylated CD68 antibody in formalin-fixed paraffin embedded colon from a normal rat. Images depict crypts and lamina propria in normal colonic mucosa. Staining for Ki-67 (unconjugated primary antibody, green), CD68 (biotinylated primary antibody, red), and DAPI (blue) as well as an overlay of the green, red, and blue channels are shown in a–d, respectively. Nuclear CD68 staining in b coincides with Ki-67-positivity in crypt nuclei in a and indicates artifactual co-localization, which results when free binding sites on tissue bound anti-mouse secondary antibody are not saturated with isotype mouse IgG1. Scale bar = 50µm

Fig. 6

Diagrammatic representation of the steps employed for performing double immunofluorescent staining with two mouse IgG1 antibodies, with each step enumerated with blue circles. a Illustration of staining protocol in which an unconjugated isotype control antibody is used to prevent interactions between tissue-bound anti-mouse secondary antibody and the biotinylated mouse antibody. First, the unconjugated primary antibody is added to the tissue to detect the first protein of interest. Second, the conjugated secondary antibody is added to the tissue to detect the unconjugated primary antibody. Third, an unconjugated isotype control is added to saturate the free Fab fragments on tissue-bound anti-mouse secondary antibody. Fourth, biotinylated primary antibody is added to the tissue detect the second protein of interest. Fifth, conjugated streptavidin is added to the tissue to detect the biotinylated primary antibody. See Online Resource 1 for an animated version of this diagram. b Illustration of staining protocol that results in artifactual colocalization (as seen in Fig. 5) caused by interactions between tissue-bound anti-mouse secondary antibody and the biotinylated mouse antibody. First, the unconjugated primary antibody is added to the tissue to detect the first protein of interest. Second, the conjugated secondary antibody is added to the tissue to detect the unconjugated primary antibody. Third, the biotinylated primary antibody is added to the tissue, where it not only binds to its target protein but also to the free arms on the secondary antibody that is bound to the tissue. Fourth, conjugated streptavidin is added to detect the biotinylated primary antibody, resulting in red fluorescence in iNOS-positive areas as well as in CD68-positive areas. See Online Resource 2 for an animated version of this diagram