Alexa fluor 568 anti rabbit igg antibody

Expression of OGA was detected by immunofluorescent staining in diploids from the 2-cell to blastocyst stage. Diploids were collected as described for detection of RNA expression. Subsequently, diploids were fixed and permeabilized with PBS-PVA that contained 0.2% (v/v) Triton X-100 (Sigma-Aldrich) and 4% (w/v) paraformaldehyde (PFA, Sigma-Aldrich) for 25 min and washed twice in PBS-PVA. Fixed diploids were stored at 4 C in PBS-PVA containing 1% (w/v) bovine serum albumin (BSA; Wako) (BPP) before use. Immunofluorescence microscopy was carried out as described below. Diploids were treated with rabbit anti-human OGA antibody (1:100; Proteintech Group, Chicago, IL, USA) in BPP at 4 C overnight. Then they were washed three times in PBS-PVA and treated with Alexa Fluor 568 anti-rabbit IgG antibody (Life Technologies) in BPP for 1 h at room temperature. After washing in PBS-PVA, diploids were mounted in ProLong Gold Antifade Mountant (Life Technologies) containing
4',6-diamidino-2-phenylindole (DAPI) and observed under an epifluorescence microscope (BX53-FL; Olympus, Tokyo, Japan).

UVB-irradiated cells were incubated for 6 h following pre-incubation with PGG for 1 h. Cells were fixed in 4% formaldehyde, permeabilized with 0.1% Triton X-100 in PBS (pH 7.4), and blocked in PBS containing 10% goat serum, 0.3 M glycine, 1% BSA, and 0.1% Tween 20 for 2 h at room temperature. For immunostaining, the cells were incubated with anti-NF-κB p65 antibody at 4°C in PBS containing 1% BSA and 0.1% Tween 20 overnight, after which they were washed, and incubated with Alexa Fluor 568 anti-rabbit IgG antibody (Molecular Probes, USA) at room temperature for 1 h. The signals were imaged using an inverted fluorescence microscope (Carl Zeiss, Germany), and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI).