Cryosections (10 μm) were used for the detection of IL-33 and F4/80 (macrophages) by immunohistochemistry following procedures outlined previously,7 (link) except that the following primary antibodies were used: IL-33 (R&D Systems) and F4/80 (Abcam, Cambridge, MA). Paraffin sections were used for the detection of mast cells by toluidine blue staining and Ly-6G (neutrophils; BD Biosciences, San Jose, CA) by immunohistochemical staining as described.7 (link), 17 (link) For quantification of cell density, cells were counted in digital images taken in high-power fields immediately adjacent to either side of the wound and area was determined using Axiovision software (Carl Zeiss Imaging Solutions, Thornwood, NY). Cell densities (cell number per mm2) were then calculated. Cell densities for the right and left sides of each wound were averaged to obtain the mean cell density for each sample.
For immunofluorescence staining of IL-33 and GFP and immunostaining for ST2, paraformaldehyde-fixed frozen sections were used in conjunction with primary antibodies specific for IL-33 and ST2 (R&D Systems) or GFP (Abcam) and appropriate Alexa Fluor-conjugated (Thermo Fisher) or biotinylated (Vector Laboratories) secondary antibodies, using standard methods. Staining was visualized using a Nikon A1Rsi resonant scanning confocal microscope.
For immunofluorescence staining of IL-33 and GFP and immunostaining for ST2, paraformaldehyde-fixed frozen sections were used in conjunction with primary antibodies specific for IL-33 and ST2 (R&D Systems) or GFP (Abcam) and appropriate Alexa Fluor-conjugated (Thermo Fisher) or biotinylated (Vector Laboratories) secondary antibodies, using standard methods. Staining was visualized using a Nikon A1Rsi resonant scanning confocal microscope.
