Mom fluorescent kit

Embryos (n=3 for each age group and genotype) were collected in ice cold PBS, fixed in 4% paraformaldehyde, and placed in sequentially higher sucrose solutions. Specimens were embedded in Tissue-Tek OCT, frozen at −80^oC and sectioned at 5μm. Immunostaining was performed using Vector Labs MOM fluorescent kit (FMK-2201) following the manufacturer’s protocol. Rhodamine phalloidin (1:100, Molecular Probes) and α-Actinin (1:400, Sigma) were used. Stained sections were photographed using Zeiss Axioskop-2 plus microscope and AxioVision Rel.4.8 software.

Dorsal skins were frozen in OCT, sectioned at 6 μm and used for Hematoxylin and Eosin staining or for immunofluorescence. Sections were fixed in 4% formaldehyde and subjected to indirect immunofluorescence. When staining with mouse mAbs, the MOM Fluorescent Kit (Vector) was used. Primary antibodies used were: mouse monoclonal Mi-2β [16G4]^{10 (link)}, rabbit polyclonal K5 (BAbCo), rabbit polyclonal K1 (BAbCo), rabbit polyclonal Loricrin (BAbCo), rabbit polyclonal K6 (BAbCo), goat polyclonal TSLP (R&D), rat monoclonal Ki67 (BD Biosciences, SolA15). Fluorescence-conjugated secondary antibodies for primary antibodies developed in rabbit, rat, or goat were obtained from Jackson ImmunoResearch Laboratories. For BrdU incorporation analysis, mice were injected i.p. with BrdU (100 μg/g body weight) (Molecular Probes). 2 h later, dorsal skin was harvested and stained with BrdU-specific antibody conjugated with Alexa Fluor 546 (Molecular Probes). DAPI was used to stain nuclei. Images were taken with the Olympus BX50 or Nikon A1 Confocal microscope.