Animal

C3H/HeJ, C57Bl/6 and Syk-/- mice at various stages of hair cycle as well as
retired breeders were purchased from Jackson Laboratories. The mice
were housed in a pathogen free barrier facility in the university premises.
Synchronized anagen was induced in the hair coat by shaving or by
plucking. Animals were administered X IFN , X LPS and X TNF and sterile
PBS via intradermal injections. Blood was obtained by retro-orbital
bleeding and stored in heparinized tubes to prevent coagulation. For
tissue harvesting, the skin was shaven, flash frozen in liquid nitrogen and
stored at -70C.

Immune Cell Isolation and Tissue Culture (Lutz)

Ex Vivo Hair Follicle Organ Culture

Immunofluorescence
Mouse skin from age matched and alopecic mice was shaved and fixed in 10%
formalin in PBS overnight followed by transfer to 70% ethanol for paraffin
embedding. Skin was also embedded in cryomatrix (Shandon, Waltham MA) and
frozen on dry ice. The frozen blocks were sectioned to a thickness of 7-8 m. The
tissue sections were further fixed in 4% paraformaldehyde or methanol and
incubated overnight for primary antibodies Rae1, panNKG2D Ligand, CD8, CD4,
neutrophils,gd TCR, NKG2D. The sections were further incubated with antigoat, rabbit or hamster, fluorescence labeled secondary antibodies, counter stained
with DAPI and mounted. Imaging was carried out using zeiss Axioscope 2 plus
microscope and Confocal (XX). Image capture and processing was done using
AxioVision , Confocal and Adobe photoshop software.
Primary Ce