Immunohistochemistry.

Immunohistochemistry for AIF1 (previously known as ionized calcium-binding adapter molecule 1, IBA1; or IFNγ-responsive transcript 1) was performed as previously validated for the detection of microglia in the brains of humans and pigs.³⁰ Briefly, formalin-fixed, paraffin-embedded tissues were sectioned (thickness, approximately 4 µm), placed on glass slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA), baked (60 °C, 60 min; Isotemp Oven, Fisher Scientific), and hydrated through a series of xylene and alcohol baths. Heat-induced epitope retrieval was performed in a citrate buffer bath at pH 6.0 (125 °C, 5 min; model DC2002, Decloaking Chamber, BioCare Medical, Concord, CA). Slides were incubated in primary rabbit antiAIF1 polyclonal antibody (dilution, 1:1000; catalog no. 019-19741, AntiAIF1/IBA1, Wako Chemicals, Richmond, VA) for 1 h room temperature. After washing, the secondary reagents (Rabbit Envision HRP System, Dako, Carpinteria, CA) were applied according to instructions. Chromogen (DAB Plus, Dako) was applied to tissues for 5 min (room temperature), followed by DAB enhancer (Dako) for 3 min, and then counterstain (Surgipath Hematoxylin, Leica Microsystems, Wetzlar, Germany) for 1 min. Finally tissues were dehydrated through a series of alcohol and xylene baths and coverslipped.

For all samples, tissue morphology was examined by 2 reviewers (KMD and DKM), one of whom is a veterinary comparative pathologist (DKM). For semiquantitative and quantitative evaluation of tissues, we followed key principles for scoring of tissues, such as evaluating immune cells.³¹ All scores were determined by the same reviewer, to decrease interobserver variation, and masking was performed by using the postexamination method.¹²

Staining (that is, brown coloration) intensity of AIF1 in the spleen was evaluated by using a semiquantitative scoring system: 0, absence of brown staining; 1, mild brown staining that did not obstruct viewing of the blue cytoplasmic counterstain; 2, moderate, distinct brown staining that partially obstructed viewing of blue cytoplasmic counterstain; and 3, strong, robust dark brown staining that completely obstructed viewing of the blue cytoplasmic counterstain. Splenic red and white pulp were graded separately for AIF1 staining intensity and compared. This staining intensity assessment was adapted from previously described approaches and methods.^1,28 For the evaluation of the hypertensive model, lung data for WKY (n = 3) and SHR (n = 3) rats were digitally collected from 2 random fields (magnification, 100×) for each animal. A masked observer ranked these images (n = 12) from least (1) to most (12) activated according to macrophage activation based on morphology. Specifically, activated alveolar macrophages were examined for larger nuclei and cytoplasm and for cytoplasmic foamy change as compared with quiescent cells; ranked scores (n = 2 per animal) were averaged for each animal for subsequent statistical analysis. Quantitative evaluation of macrophage diameter was made for each digital image, and these results were pooled for each animal. Scoring data were analyzed (Prism, GraphPad Software, San Diego, CA) by using a Wilcoxon matched-pairs signed rank test for splenic samples and unpaired t-tests for lung samples. Results were considered significant at a P value less than 0.05.