Biotinylated goat anti rat

Lungs from the four groups [RA, n=4; RA & CPAP, n=4; 50% O₂, n=6; and 50% O₂ & CPAP, n=6] were inflation-fixed [25 cm H₂O] for 10 minutes with 10% neutral-buffered formalin. The 5 μm paraffin embedded tissue was treated with 3% H₂O₂ after antigen retrieval. Lung sections were incubated 24 hours at 4μC with rat anti-mouse Mac3 monoclonal antibody [1:500; BD Pharmingen, San Jose, CA]. The secondary antibody was biotinylated goat anti-rat [1:500 Jackson immunoResearch West Grove, PA, USA], followed by ABC reagent incubation and visualization by diaminobenzidine [Vectastain, Vector Laboratories, Burlingame, CA, USA]. Finally, lung sections were dehydrated and mounted with permount. Primary antibody was omitted for negative controls. All images were obtained using a Rolera XR CCD camera [Q-Imaging, Canada] mounted on a Leica DMLB microscope [Leica Microsystems, Germany]. Images were analyzed using research-based digital image analysis software [Image-pro Plus 7.0, Media Cybernetics, Silver Spring, MD]. Numbers of labeled macrophages were counted in five random fields at 20x magnification from 4-6 animals per group.

At the end of the study, mice were sacrificed with deep anesthesia (3 g/kg urethane) followed by transcardiac perfusion using approximately 10 ml ice-cold 1X PBS (diluted from 10X stock Boston Bioproducts, Inc) followed by approximately 12 ml ice-cold 4% paraformaldehyde (PFA). The brain was post-fixed overnight in 4% PFA and then placed in 30% sucrose until sectioning. Brains were sectioned (40 microns) using a cryostat and floated in 1X PBS prior to immunostaining. All solutions were diluted in PBS with 0.3% Triton-X (PBST), and sections were rinsed in PBS 3 times between each step. Sections were first incubated in a 1% hydrogen peroxide solution for 60 min followed by overnight incubation in primary antibody (1:20,000 rat anti-mCherry, Life Technologies #M11217) diluted in blocking solution (5% normal goat serum in PBST). Sections were then placed in secondary antibody (1:1000 biotinylated goat anti-rat, Jackson Immuno #112–066-003) for 30 min and 1:1000 ABC (Vector Elite Kit, Vector Labs) for 60 min. All rinses and incubations occurred on a rocker at room temperature. Finally, sections were incubated in a solution of 0.05% 3,3’-diaminobenzidine and 0.015% hydrogen peroxide for 10 min.

Immunohistochemistry and histology was performed by the University of Washington Histopathology Shared Resource. Five-micron sections were cut, deparaffinized and rehydrated in Dako Wash Buffer (Dako, Carpinteria, CA). Slides were antigen retrieved in a Black and Decker steamer for 20 minutes in preheated Trilogy buffer (Cell Marque, Hot Springs AZ) and cooled for 20 minutes. Slides were rinsed 3 times in wash buffer and all subsequent staining steps were performed at room temperature using the Dako Autostainer. Endogenous peroxide activity was blocked using 3% H₂O₂ for 8 minutes followed by protein blocking. Slides were blocked in 15% goat serum and 5% canine serum in TBS containing 1% BSA for 10 minutes. Antibodies were used at 10 ug/ml and incubated for 60 minutes and were detected using biotinylated goat anti-rat (112-065-167, Jackson ImmunoResearch) at 1:200 for 30 minutes followed by Vector Elite ABC. The staining for all slides was visualized with 3,3’-diaminobenzidine (Dako,Carpinteria, CA) for 8 minutes, and the sections were counter-stained with hematoxylin (Dako, Carpinteria, CA ) for 2 minutes. Concentration matched isotype control slides were run for each tissue sample (Jackson ImmunoResearch Laboratories).