Rhodamine red x goat anti rabbit igg h l

Two days after transfection with the MED25 mammalian expression vector, the monolayer of adherent 293T cells on the plate was washed once with PBS and then fixed with PBS containing 4% paraformaldehyde (Sigma Aldrich) at room temperature for 10 min. Next, the cells were permeabilized using PBS containing 0.25% Triton X-100 at room temperature for 8 min and then blocked with PBS containing 5% Fetal bovine serum (FBS, Gibco, Waltham, MA, USA) at room temperature for 10 min. After washing once with PBS for 5 min, primary antibodies the same as the ones used for Western blot above were added separately at 60 μg/mL and incubated at 37 °C for 90 min. After washing three times with PBS, the fluorescent dye labeled secondary antibody Rhodamine Red-X Goat Anti-Rabbit IgG (H+L) and Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) (Invitrogen) were added for incubation at 37 °C for 40 min according to the user manual. Following another three washes, the cells were finally stained with the DAPI nucleic acid stain (Invitrogen) at room temperature for 5 min as the user manual described. After staining, the plate was washed once and sealed with 50% glycerol. Fluorescence signals were observed using the IX71 inverted research microscope (OLYMPUS) at 400× magnification.

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.