Alexa fluor 488 goat anti mouse igg h l secondary antibody

Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS for 5 min at room temperature. Cells were stained with the primary antibodies for 1 h and secondary antibodies for 45 min each at room temperature. DNA was counterstained with DAPI. Primary antibodies included the following: TP63 (1:100, CM163A; Biocare Medical), TTF1 (1:200, ab76013; Abcam), SOX2 (1:200, 09-0024; ReproCELL USA Inc., Beltsville, MD, USA), PE-conjugated human EpCAM (1:200, 12-9326-42; Thermo Fisher Scientific), MUC5AC (1:200, MA1-38223; Thermo Fisher Scientific), APC-conjugated human CD47 (1:20, 323123; Biolegend), PE-conjugated human CD26 (1:20, 302705; Biolegend, San Diego, CA, USA), Alexa Fluor 647-conjugated human Podoplanin (1:200, 337007; Biolegend), and Alexa Fluor 488-conjugated human SP-C (1:200, sc-518029AF488, Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies included Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody (1:200, A11029; Thermo Fisher Scientific), Alexa Fluor 568 donkey anti-rabbit IgG (H+L) secondary antibody (A10042; Thermo Fisher Scientific), and Alexa Fluor 594 goat anti-mouse IgG (H + L) secondary antibody (A11032; Thermo Fisher Scientific).

For protein staining, cells were seeded onto coverslips, stimulated with anti-CD3 for the indicated times, and then fixed with 4% paraformaldehyde for 30 min at room temperature followed by permeabilization with 0.1% Triton X-100 for 5 min and blocking with 10% FBS for 30 min at room temperature. Cells were then incubated with primary antibodies in 5% FBS at 4 °C overnight. After washing three times (10 min each) with PBS, cells were incubated with fluorescent secondary antibody conjugates at 37 °C for 30 min followed by staining with Hoechst 33342 at room temperature for 10 min. Antibodies are used as following: Rabbit anti-ORP4L (Sigma-Aldrich, 1:200), mouse anti-OCRL (Santa Cruz, 1:50), Alexa Fluor 488-Goat Anti-Mouse IgG (H + L) secondary antibody (Thermo Fisher Scientific, 1:200), and Alexa Fluor 546 Goat Anti-Rabbit IgG (H + L) secondary antibody (Thermo Fisher Scientific, 1:200). The specimens were analyzed using Olympus FV3000 laser-scanning confocal microscope system.

An Annexin V-FITC kit was obtained from BD Biosciences. Antibodies against ERK5, phospho-ERK5, ERK1/2, phospho-ERK1/2, phospho-checkpoint kinase 1 (Chk1), phospho-checkpoint kinase 2 (Chk2), phosphor-ATM kinase, phosphor-ATR protein, cleaved caspase-9, cleaved caspase-8, cleaved caspase-3, Cyclin B1, Cdc-2, and Cdc25C were purchased from Cell Signaling Technologies. Antibodies against p53, α-tubulin, β-actin, and cleaved poly (ADP-ribose) polymerase (PARP), and the ERK5 inhibitor XMD8-92 were purchased from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG antibodies were also obtained from Santa Cruz Biotechnology. Anti-H2A histone family, member X (H2AX, Ser139) was provided by Millipore (Billerica, MA, USA). Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody was purchased from Invitrogen (Carlsbad, CA, USA). Anti-CD31 antibody was obtained from Abcam (Cambridge, MA, USA). Sources of other materials are noted accordingly in the study.

HEK293T cells were transfected with various eqTHN-expressing plasmids with FLAG peptide in extracellular domain for 24 h. After fixation with 4% paraformaldehyde, the cells were incubated with the mouse anti-FLAG antibody (Sigma) at 1:1000 dilution for 1 h. After washing, the cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (H + L) secondary antibody (Invitrogen, Waltham, MA, USA) at 1:1000 dilution for 1 h. The mean fluorescence intensity of eqTHN localization on the cell surface was then determined by flow cytometry.

HeLa cells were seeded at 1 x 10⁵ cells per well in 24-well plates containing glass cover slips. On the following day, cells were transfected with the pRINES/122T plasmid and incubated with Cumate for 48 hours. Cells were fixed and permeabilized with cold methanol for 20 min, washed in PBS, incubated with the hNIS antibody (1/100 dilution) for 40 min at room temperature followed by an Alexa Fluor 488 goat anti-mouse IgG (H + L) secondary antibody (Invitrogen) for 30 min at room temperature and washed again with PBS. Then, the cover slips were mounted on slides using Vectashield mounting medium (Vector Laboratories, USA) for confocal microscopy observation [21 (link)]. Slides were analysed using an LSM 510 META scanning device coupled to an Axiovert 200 microscope (Carl Zeiss, Germany), using the 63 x high numerical-aperture oil immersion objective lenses. Image sizes were set to 512 × 512 pixels. Z-stack images were acquired randomly for each condition, and representative images from the focal plane of the nucleus were analysed by Image J v1.4.9 software. Regions of Interest (ROIs) were selected using the polygon select tool of the software and the mean fluorescence intensity (MFI) determined. For quantification studies, ratios between MFI expressed in pixels and the selected area of ROIs expressed as relative unit of surface (RUS) were calculated and plotted on graphs.

Cells seeded on coverslips were fixed for 10 min in 4% paraformaldehyde in 10 mmol/L piperazine- N,N′-bis (2-ethanesulfonic acid) (PIPES), pH 6.8, 10 mmol/L NaCl, 300 mmol/L sucrose, 3 mmol/L MgCl₂, and 2 mmol/L EDTA. Cells were permeabilized for 10 min in Tris-buffered saline (TBS) with 0.75% Triton X-100 and blocked for 10 min in 5% bovine serum albumin and 0.1% Triton X-100 in TBS. Cells were then incubated with antibodies against BARF1 (MAb 6F4, 1:100) [5 (link)] and SMAD4 (sc-7966, 1:200 Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody (Invitrogen). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/mL). Stained cells were visualized on a Zeiss Observer.Z1 fluorescence microscope or a Zeiss LSM710 confocal system (Carl Zeiss Meditec, Jena, Germany).