Alexa fluor 488 goat anti rat igg

Skin samples were fixed in 4% paraformaldehyde (PFA) in PBS overnight and submitted to the Johns Hopkins Oncology Tissue Services where they were embedded in paraffin, sliced into 4 μm sections, mounted and stained with hematoxylin and eosin (H&E). Gram stain was performed using the Gram Staining Kit (Sigma Aldrich) according to manufacturer’s instructions. For immunofluorescence labeling, paraffin sections were deparaffinized, underwent heat-mediated antigen retrieval in either Trilogy buffer (Cell Marque) or Tris-EDTA buffer (10mM Tris Base, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0), blocked in blocking buffer (PBS with 10% goat serum) for 1 hour at room temperature, and then incubated at 4° C overnight with 1 μg/mL rabbit anti-mBD14, rat anti-mouse Ly6G or rabbit anti-S.aureus primary antibodies in blocking buffer. The next day, sections were incubated for 1 hour at room temperature with 1 μg/mL of either AlexaFluor-488 goat anti-rabbit IgG (ThermoFisher, for mBD14 staining), AlexaFluor-488 goat anti-rat IgG (ThermoFisher, for Ly6G staining) or AlexaFluor-594 goat anti-rabbit IgG (ThermoFisher, for S. aureus staining). All slides were washed and mounted with Fluoromount-G with DAPI (ThermoFisher). All microscopy were performed on a Keyence BZ-X710 microscope (Keyence) and analyzed with Image J (National Institutes of Health Research Services Branch).