Fluoromount g mounting medium with dapi

For immunohistochemical staining of GFP in the CA1 area of the mouse brain, mice were anesthetized with pentobarbital (50 mg/kg, i.p.) and perfused with ice-cold PBS, followed by 4% paraformaldehyde. Brains were removed and post-fixed in 20% sucrose/4% paraformaldehyde solution for 20–48 h. Brains were then frozen, cut into 30-μm sections on a cryostat and mounted on gelatin-coated slides. Brain sections were rinsed with 1 X PBS for 10 min and permeabilized with pre-cold EtOH/CH3COOH (95%:5%) for 10 min, followed by 1 X PBS for 10 min for three times. The sections were pre-incubated in a blocking solution containing 3% bovine serum albumin (BSA) and 0.1% Triton X-100 in 1 X TBS for 1 h, followed by 1 X PBS for 10 min for three times. For examination of lentiviral vector transduction, brain sections containing the CA1 area were prepared for visualization of GFP (green) fluorescence. For immunofluorescence detection of the nucleus, tissue sections were added with 20 μl of the Fluoromount-G mounting medium with DAPI (SouthernBiotech, Birmingham, AL). Photomicrographs were taken using a Zeiss LSM700 confocal microscope.

Skin biopsies of ACD patients and normal skin biopsies were embedded in paraffin, cut into 4 μm thick sections, and heated for 20 min at 63 °C. The samples were stained using a BOND Autostainer and included dewaxing, pre-treatment with a buffer pH 9 for 20 min at 95 °C, and sequential double staining. Skin biopsies from positive patch test reactions and normal skin biopsies were placed in an optimal cutting temperature (OCT) compound, snap-frozen, and stored at −80 °C. Samples were cut into 6 μm cryo-sections, fixed with acetone at 4 °C for 10 min, and blocked with normal goat serum (1:50) (Dako) at room temperature for 15 min. Primary antibodies were added at room temperature for 60 min, followed by washing with TBS-Saponin 0.1%. Secondary antibodies were also added at room temperature for 60 min, followed by washing with TBS-Saponin 0.1%. Slides were mounted using Fluoromount-G™ Mounting Medium with DAPI (Southern Biotech). Immunofluorescence images were acquired on an Eclipse Microscope (Nikon) using the NIS Elements Imaging v. 4.2 software (Nikon). All antibodies used for Immunofluorescence are listed in Supplementary Table S1.