Ds ri1 digital camera

For immunofluorescence analysis of skin biopsies, 5 μm paraffin-embedded sections were kept overnight at 37°C and de-paraffinized using xylene/ethanol. Antigen retrieval was done with 0.01M citrate buffer, pH 6.0 (Invitrogen, Carlsbad, CA) in a microwave for 25 min. Sections were blocked with 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 30 min at room temperature. Primary antibodies used: rabbit anti-TSPEAR primary antibody (Abcam, Cambridge, MA, USA, 1:200 dilution); goat anti-NOTCH primary antibody (Santa Cruz, Dallas, TX, USA, 1:75 dilution). Both antibodies were diluted in 2% BSA PBS and incubated overnight at 4°C. Rhodamine Red-X goat anti rabbit IgG (H+L) (Life Technologies/Invitrogen) and Alexa Fluor 568 donkey anti goat IgG (H+L) (Thermo Fisher Scientific) were used as a secondary antibody and were diluted 1:200 with 2% BSA in PBS followed by incubation for 45 min at room temperature. Coverslips were mounted in DAPI Fluoromount-G (Southern Biotechnologies, Birmingham, AL). Negative controls consisted of slides processed similarly while omitting the primary antibody. As a positive control for TSPEAR staining, we used normal placenta tissue[42 (link)]. Specimens were examined using either a Nikon 50I microscope connected to DS-RI1 digital camera or a Zeiss LSM700 confocal microscope for fluorescence image acquisition.