Alexa fluor 488 conjugated goat anti rabbit igg h l secondary antibody

Tissue sections were prepared from the inner ears of wild-type, Slc26a4^{919-2A>G/919-2A>G}, and Slc26a4^T721M/T721M mice. The sections were mounted on silane-coated glass slides, deparaffinized in xylene, and rehydrated in ethanol. Tissues were stained with a 1:1000 dilution of rabbit anti-pendrin antibody kindly provided by Dr. Jinsei Jung, Yonsei University College of Medicine, Seoul, Republic of Korea. This was followed by exposure to DAPI (1:5000) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:200; Thermo Fisher Scientific). After incubation, the slides were washed with PBS and mounted with ProLong Antifade kit at 25 °C. Images were obtained using the aforementioned LSM 880 laser scanning confocal microscope^{10 (link)}.

The localization of FtsZ in Synechococcus WT and mutant strains was evaluated by immunofluorescence using a modified protocol from Heinz et al.^{101 (link)}. In contrast, cells were lysed in 50 mM Tris-HCl pH 7.4, 10 mM EDTA and 0.2 mg ml⁻¹ lysozyme for 30 min at 37 °C and samples were blocked in 1x Roti®-ImmunoBlock (Carl Roth) in PBS supplemented with 0.05% Tween 20. Samples were incubated with rabbit anti-FtsZ primary antibody (Agrisera; raised against Anabaena FtsZ; 1:250 diluted) in blocking buffer followed by incubation with 7.5 µg ml⁻¹ Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) secondary antibody (Thermo Fischer Scientific) in blocking buffer. Before microscopy, cells were stained with 10 µg ml⁻¹ DAPI (final concentration) in PBS.

HUVECs were washed with cold PBS, fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, and blocked with 10% normal goat serum (Cell Signaling Technology) in PBS for 1 h. Cells were incubated with rabbit anti-LC3B antibody (1:100 dilution, Cell Signaling Technology), mouse caspase-8 antibody (1:50, dilution, Santa Cruz Biotechnology Inc.), and guinea pig anti-p62 antibody (1:100 dilution, Progen, Wayne, PA, USA) in 1% bovine serum albumin (BSA)-containing PBS overnight at 4 °C. Cells were then washed with 0.5% Tween-20-containing PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H&L) secondary antibody (Invitrogen), Alexa Fluor 568-conjugated goat anti-mouse IgG (H&L) secondary antibody (Invitrogen), and Alexa Fluor 405-conjugated goat anti-guinea pig IgG (H&L) secondary antibodies (abcam). After three washes with 0.5% Tween-20 containing PBS, cells were mounted using ProLong^® Gold antifade reagent (Invitrogen) and immediately analyzed under a confocal microscope (FVS3000-ORS, Olympus Corporation). The representative image was presented from three independent experiments.

Cells grown on glass coverslips in a 12-well plate were fixed in 4% paraformaldehyde (PFA) for 15 min, permeabilized with Triton X-100 for 5 min, and incubated in sodium borohydride for 10 min. They were then incubated with rabbit anti-geminin antibody (1:200) in 1x PBS containing 1% bovine serum albumin (BSA) for 1 h at room temperature (RT). Cells were washed 5 times with 1x PBS, incubated with Alexa Fluor 488–conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:200; Invitrogen, Carlsbad, CA, United States ) in 1x PBS containing 1% BSA for 45 min at RT in the dark, followed by washing twice with 1x PBS. DNA was visualized by staining with 4’,6-diamidino-2-phenylindole (1:5,000 in water; Invitrogen). Cells were visualized with a laser scanning confocal microscope (LSM 710; CarlZeiss, Wetzlar, Germany).

LLC cells at a density of 1×10⁵ cells/well were cultured on coverslips in 6-well plates. After 24h of incubation, the NC, HF, RT, RT+HF, and RT+SB cells were treated as indicated, and the cells were further incubated for 48h. Cells were fixed in 4% paraformaldehyde for 15 min at 4°C, permeabilized with 1% Triton-100 for 30 min at room temperature. Coverslips were incubated in primary antibodies against Cytokeratin (#9384, 1:1000, CST) or Vimentin (#5741, 1:1000, CST) overnight at 4°C, followed by Alexa fluor 488-conjugated goat anti-rabbit IgG (H + L) secondary antibody (Invitrogen) for 1h at 37°C. Cells were washed and nuclei were counterstained with 1 mg/ml DAPI. Images were acquired with an OLYMPUS BX61 confocal microscope with a 40×objective. The results from at least 3 different experiments were averaged.

Day 7 MCF7–MSC co-cultures were fixed with methanol/acetone 1:1 (vol/vol) for 30 minutes at −20 °C. After fixation, cells were dried for 15 minutes and rehydrated with PBS for 15 minutes. Cells were blocked with 2 % BSA (Sigma) for 1 hour, followed by incubating with rabbit primary antibodies against Ki67 (1:200, ab15580; Abcam, Cambridge, MA, USA) or Proliferating cell nuclear antigen (PCNA) (1:100, ab2426; Abcam) in blocking solution at 4 °C overnight. After removal of primary antibodies, cells were washed three times with PBS, and then Alexa Fluor® 488 conjugated goat anti-rabbit IgG (H + L) secondary antibody (1/1000, A11008; Lifetechnologies, Carlsbad, CA, USA) was added and incubated for 2 hours at room temperature. Cells were washed three times with PBS and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) nuclear dye, mounted on slides in CC/Mount (C9368; Sigma), and were observed under a Nikon®ECLIPSE Ti fluorescence microscope.