Fitc conjugated goat anti rabbit igg

The ureter tissues were fixed with 4% paraformaldehyde. Then, the ureter tissues were embedded in paraffin, cut into sections, and prepared for immunohistochemistry. After quenching endogenous peroxidase with 3% H₂O₂, the tissue slides were incubated with 1% normal rabbit serum to minimize nonspecific binding. Next, the slides were incubated with rabbit polyclonal anti-Piezo1 antibody (1:200, Proteintech, United States) at 37°C for 1 h in a dark, humidified box. After washing with PBS, the slides were incubated with FITC-conjugated goat anti-rabbit IgG (1:100, Abcam, United Kingdom) for 30 min. After washing with PBS, the slides were then incubated with 100 ng/mL DAPI for 10 min. After washing with PBS, the slides were embedded in 50% glycerol. Tissues were visualized using a Leica microscope (Leica, Germany). Immunohistochemical staining mean optical density (MOD) values to quantify staining results. Representative images were obtained with a digital camera and then processed with Image-Pro Plus 6.0 software.

BslA_(260–652) protein was added to the pretreated cells (25 μg of protein/1 × 10⁶ cells) and incubated for 1 h at 4 °C. Cells were washed three times with PBS, and suspended in PBS containing 3 % FCS and BslA_(260–652)-specific antiserum (1:100, v/v) and incubated on ice for 1.5 h. After washing 3X with PBS, the cells were incubated with FITC-conjugated goat anti-rabbit IgG (1:1000, v/v; Abcam) on ice for 1 h. Then the cells were stained with 0.5 mg/ml of 7-AAD (KeyGen Biotech) for 10 min and the FITC-labeled cells (7-AAD negative, live) were examined with FACSCalibur (BD Biosciences, USA).

ARPE-19 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Dulbecco’s Modified Eagle’s Medium: nutrient mixture F12 (hereafter named DMEM:F12), fetal bovine serum (FBS), bovine serum albumin (BSA), trypsin-EDTA, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium bicarbonate, gentamycin, phosphate-buffered saline (PBS), penicillin, streptomycin, 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), Tween-20 and PAP pen were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein isothiocyanate (FITC)-labeled annexin V (to bind PS), annexin V-binding buffer containing 10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl₂, were purchased from Becton Dickinson Biosciences (BD), Belgium. Minimum essential medium (MEM) was purchased from Invitrogen (Carlsbad, CA, USA). Pipettes, 25 cm² flasks, 15 mL and 50 mL centrifugation tubes, 1 L glass bottles, and pipette tips were supplied by VWR International (West Chester, PA, USA). Vacuum filtration rapid filter mix was supplied by BioNordika (Oslo, Norway). Mouse anti-RPE65, rabbit anti-occludin, FITC-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG antibodies were obtained from Abcam (Cambridge, UK). Mouse anti-ZO-1 and Alexa Fluor 568 phalloidin were purchased from Life Technologies (Carlsbad, CA, USA).

HSYA (C27H32O16; MW, 612.53; 98% Purity; Solarbio, Beijing, China), lecithin (Aladdin, Shanghai, China), bleomycin (BLM Biofeng, Shanghai, China), pirfenidone (Aladdin, Shanghai, China), ELISA kits (for TGF-β1 and IL-6; Cloud-Clone, Wuhan, China), 4% paraformaldehyde (Solarbio, Beijing, China), hematoxylin and eosin staining kit (beyotime, Shanghai, China), Weigert hematoxylin staining solution (Solarbio, Beijing, China), Ponceau S and Magenta staining solution (Solarbio, Beijing, China), phosphomolybdic acid solution (Solarbio, Beijing, China), Aniline blue solution (Solarbio, Beijing, China), DAPI (Solarbio, Beijing, China), RIPA buffer (Thermo Fisher Scientific, Waltham, USA), antibodies (for p38, p-p38, Smad3, collagen I, β-actin (Servicebio, Wuhan, China), anti-mouse IgG (Abcam, Cambridge, UK), anti-rabbit IgG (Abcam, Cambridge, UK)), BCA Protein Assay Reagent Kit (beyotime, Shanghai, China), and primary antibodies (CD45, α-SMA, and collagen I (Abcam, Cambridge, UK)), FITC-conjugated goat anti-rabbit IgG (Abcam, Cambridge, UK), CY3-conjugated goat anti-rat IgG (Abcam, Cambridge, UK), and CY5-conjugated goat anti-mouse IgG (Abcam, Cambridge, UK), were purchased.

To assay the expression of a specific marker in keratinocytes and in vitro differentiated hASCs, immunofluorescence staining was performed following the established method [49 (link)]. Similar to the adhesion assay, the samples (polyester membranes and CSSs) with cells in 96-well plates (REF# 3599, Corning) also underwent these processes such as being rinsed, fixed, and penetrated. After being blocked with 1% BSA at room temperature for 1 h, the samples were incubated with primary anti-K14 (mouse monoclonal; 1:100, REF# 7800, Abcam) at 4 °C overnight, followed by secondary antibody including FITC-conjugated goat anti-rabbit IgG (mouse monoclonal; 1:100, REF# 6785, Abcam) solution for 1 h at room temperature in a dark place. Nuclei were labeled by DAPI for 5 min and stained samples were kept at 4 °C after two washing steps. The cells were inspected and photographed using a laser scanning confocal microscopy (LSCM; SP8, Leica) at 493/528 nm. The average fluorescence intensity per unit area was calculated using Image-Pro Plus software (version 6.0). Five low-magnification (× 20) images of the individual samples in each group were used for calculation.

Cell suspensions were air dried on poly-l-lysine-coated glass slides before fixation in 4% paraformaldehyde for 15 min at room temperature. The cells were then washed three times in PBS and blocked with PBS containing 10% goat serum and 0.1% Triton X-100 for 1 h. Anti-Notch2 (Santa Cruz Technology, sc-5545) antibody was diluted in 0.1% Triton X-100-PBS and was used to incubate cells overnight at 4°C. Cells were then washed three times in PBS followed by incubation with FITC-conjugated goat anti-rabbit IgG (Abcam, ab6717) for 1 h at room temperature. Cells were then washed two times in PBS and mounted with ProLong^® Gold Antifade Mountant (Thermo Fisher Scientific, P36930). Cells were observed on an Olympus BX51 fluorescence microscope.

MTT method was performed to test the cytotoxicity of the proteins. HeLa cells were treated with MsrA or PEP-1-MsrA (0–18 μM) for 72 h. To determine the transduction efficiency of PEP-1-MsrA, peritoneal macrophages were treated with various concentrations of PEP-1-MsrA or MsrA protein (0.5–8 μM) for 1 h. Then cells were washed with PBS and harvested for Western blot analysis. The cells were also treated with 1 μM of proteins for various times (5 min–24 h) to examine the stability of PEP-1-MsrA by Western blot analysis.
We further detected the intracellular distribution of transduced protein using an immunofluorescence assay. Briefly, peritoneal macrophages were seeded on coverslips and treated with selected concentrations of PEP-1-MsrA or MsrA protein (1–4 μM) for 1 h, and then washed with PBS at least three times and fixed with 4 % paraformaldehyde for 10 min at 37 °C. The cells were incubated with the primary antibody (anti-MsrA antibody, Abcam, USA) overnight at 4 °C, then with FITC-conjugated goat anti-rabbit IgG (1:200) for 2 h at room temperature in the dark. Nuclei were stained with 300 nM DAPI (Sigma-Aldrich, USA) for 15 min at 37 °C. The intracellular localization of MsrA protein was analyzed with confocal microscopy (Olympus FluoView FV1000, Japan).