Alexa fluor 647 conjugated goat anti rabbit igg secondary antibody

As previously described (Raffay et al., 2016 (link)), after terminal anesthesia by ketamine/xylazine overdose, animals were perfused in-situ by right ventricular puncture with PBS (pH 7.4) then 10% formalin, cannulated by tracheostomy and lungs inflation-fixed with 10% formalin at 25 cm H₂O. The trachea was ligated and the lungs were post-fixed in 10% formalin at 4° C for >24 hours. Tissue was paraffin embedded and 5 μm lung sections were processed for histology or immunostaining. Hematoxylin/Eosin and Masson’s Trichrome staining were performed by the CWRU Tissue Resource Core. Immunofluorescent tissue visualization for EpCAM and MPO by confocal microscopy was performed essentially as previously described (Johnson et al., 2015 (link), 2018 (link)). Tissue slices on slides were deparaffinized and rehydrated prior to antigen retrieval in boiling citrate. Slides were blocked with serum and incubated overnight. Slides were washed and incubated with AlexaFluor 488-conjugated rat anti-mouse EpCAM (BioLegend) and rabbit anti-mouse MPO (Abcam) antibodies. Slides were then washed and stained with AlexaFluor 647-conjugated goat anti-rabbit IgG secondary antibody (Abcam). Slides were imaged on a Leica SP5 Scanning Confocal Microscope.