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 TNFα 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 -70C.

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
µm. 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 anti- goat, 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