Alexa fluor 488 rabbit anti mouse igg

Unless otherwise stated, chemicals were used without further purification. Poly-L-lysine hydrobromide (PLL; M_w 30–70 kDa), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) ≥ 98.0% and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich. Hyaluronic acid (HA; M_w 6.4 kDa, 752 kDa and 1500 kDa) was purchased from Lifecore. We used Anti-CD44 antibody [KM201] (ab25340) and Mouse IgG1 Kappa [MOPC-21]-Isotype 1 (ab18443) from Abcam for the CD44 blocking. Recombinant Human CD44 His tag protein was purchased from Biorbyt. Phalloidin–tetramethylrhodamine B isothiocyanate (phalloidin–TRITC), 4,6-diamidino-2-phenyindole, dilactate (DAPI) from Sigma-Aldrich and the monoclonal antibody to CD44 from Acris-Antibodies were used for immunostaining. The secondary antibody, Alexa Fluor 488 Rabbit Anti-Mouse IgG, was obtained from Invitrogen. The primary antibody Cortactin (H-191), sc-11408, was obtained from Santa Cruz Biotechnology.

HEK-293T, MDCK, and human cervical HeLa cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37°C/5% CO₂. Infectious wild-type and NES mutant viruses (produced by reverse genetics, as described below) were derived from influenza A/WSN/33. A polyclonal antibody (Ab) against influenza A/WSN/33 virus proteins (anti-WSN Ab: a kind gift from Dr. Kazufumi Shimizu, Nihon University School of Medicine) was used for Western blot analysis together with horseradish-peroxidase (HRP)-conjugated goat anti-rabbit IgG (Amersham Bioscience). Monoclonal antibodies (MAbs) against M1 (Santa Cruz), NS1 (Santa Cruz), CRM1 (BD Biosciences), and β-actin (Sigma), and an HRP-conjugated goat anti-mouse IgG (Amersham Bioscience), were also used. Anti-NS1 MAb (Santa Cruz), anti-NS2 Ab (GeneTex), anti-NP MAb (Santa Cruz), anti-M1 MAb (Abcam), Alexa Fluor 488 rabbit anti-mouse IgG (Invitrogen), and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) were used for immunofluorescence staining.

The frozen brains were fixed with 30% sucrose in 0.5% paraformaldehyde solution overnight before serially sectioning into 40 μm along the coronal plane to get the whole dentate gyrus (bregma point -2.3 to -6.3 mm) [42 ] using a cryostat (Cryostat Series HM550 Microm international, A.S. Science Co., Ltd., Walldorf, Germany) and collected into 24-well plates containing the cryoprotective buffer. Sections were incubated in blocking buffer (20% normal goat serum (Abcam, Cambridge, UK) in PBS containing 0.5% Triton X-100) for 1 h and incubated with an anti-p21 (1 : 100, Santa Cruz Biotechnology, TX, USA) at 4°C overnight. The next day, sections were washed with buffer (PBS containing 0.3% Triton X-100), followed by incubating with an Alexa Fluor 488 rabbit anti-mouse IgG (1 : 300, Invitrogen, San Diego, CA, USA) for 1 h, and were labeled with propidium iodide (PI) (1 : 6000, Sigma-Aldrich, St. Louis, MO, USA). Nine sections from every 8th brain section of the entire length of the dentate gyrus were selected from each rat for staining [43 (link)]. The p21-positive cells within the three cells of the internal rim of the dentate gyrus were scored, and a total positive cell count of nine sections from each rat were multiplied by 8 for quantification. Fluorescence images were taken at 10× and 40× magnifications using a fluorescence microscope (Nikon ECLIPSE 80i, Melville, NY, USA).

U-937 cells were seeded in ibiTreat µ-Slide 8 Well chambers slide (Ibidi; Germany, Cat#80826) (200 000 cells/well) after being subjected to lentiviral transductions. Cells were fixed with 4% (w/v) paraformaldehyde (Thermo Fisher Scientific; Cat#28906), permeabilized with Triton X-100 0.2% (Sigma-Aldrich; Cat#X100), and incubated with anti-FLAG mouse monoclonal primary antibody (1:500; Merck; Cat#F1804) and Alexa Fluor 488 rabbit-anti-mouse IgG (1:1000; Invitrogen; Cat# A27023) secondary antibody. Cells were washed with PBS between steps and incubated with DAPI (5 min; 1:5000; Invitrogen; D1306) before mounting with ibidi mounting medium (Ibidi; Cat#50001). Fluorescence images were acquired using a Leica-SP8 confocal microscope with a HC PL APO CS2 40/1.10 water objective and analysed using the Bitplane Imaris software (Imaris, Zurich, Switzerland).

On Day 3 of culture, the cells on chambered glasses were fixed for 10 min with 10% formalin (Sigma, St. Louis, MO, USA) and washed three times with phosphate buffered saline (PBS; Sigma, St. Louis, MO, USA). The cells were permeabilized by 0.1% Triton X-100 for 5 min and washed with PBS three times. To prevent nonspecific binding of the antibody, 1% bovine serum albumin (BSA; MP biomedicals, Singapore) was treated for 30 min at room temperature. Then the cells were treated with the Lamin A/C antibody (Santa Cruz Biotehcnology, Dallas, TX, USA) diluted in the ratio of 1:100 in 1% BSA at room temperature for 1 h. After washing three times with PBS, the secondary antibody was treated at room temperature for 1 h. As a secondary antibody, Alexa Fluor 488 rabbit anti-mouse IgG (invitrogen, USA) diluted in the ratio of 1:500 in 1% BSA was used. After washing with PBS, the cell nuclei were stained for 7 min using Hoechst 33258 (Thermo Fisher, Waltham, MA, USA). Confocal microscopy (Carl zeiss, Jena, Germany) was used to confirm fluorescent signals of Lamin A/C and cell nuclei with ×40 magnification.