For immunohistochemical fluorescence analysis, cell-loaded scaffolds were cultured in 24-well plates for 1 or 7 d and fixed in 4% paraformaldehyde. The cell-loaded scaffolds were washed three times before being incubated with 0.1% Triton X-100 at room temperature for 30 min and then incubated with 5% bovine serum albumin (BSA, Gibco, Grand Island, NY) at 37°C for 1 h. The cell-loaded scaffolds were washed three times before being incubated with Rhodamine Phalloidin (Beyotime Institute of Biotechnology, Jiangsu, China) at 1 : 200 dilution overnight at 4°C and then incubated with DAPI for 15 min. Immunoreactivity was assessed using a laser confocal microscope.
Rhodamine phalloidin
Manufactured by Beyotime
Sourced in China
Rhodamine phalloidin is a fluorescent stain used to visualize actin filaments in cells. It binds specifically to F-actin, allowing for the detection and localization of the actin cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (4 mentions)
Anti vinculin, by Proteintech (2 mentions)
Fv1200, by Olympus (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Lactate dehydrogenase ldh release assay kit, by Beyotime (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Mrc 5, by American Type Culture Collection (1 mentions)
H3122, by American Type Culture Collection (1 mentions)
Il 10, by Wuhan Servicebio Technology (1 mentions)
Goat anti mouse igg, by Wuhan Servicebio Technology (1 mentions)
Anti pdpn, by Proteintech (1 mentions)
Goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Anti ctsk, by Abcam (1 mentions)
Ckx53, by Nikon (1 mentions)
Il 1β, by Wuhan Servicebio Technology (1 mentions)
Cd11b, by Wuhan Servicebio Technology (1 mentions)
Confocal microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
8 protocols using rhodamine phalloidin
1
Immunohistochemical Fluorescence Analysis of Cell-Loaded Scaffolds
2
Visualization of Nano-TiO2 Internalization in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescein isothiocyanate (FITC)–nano-TiO2 was produced according to a previously reported method.40 (link) Briefly, FITC-nano-TiO2 was prepared and added to cells at various concentrations (0, 10, or 40 µg/mL) for 24 hours after further selective staining of the cell skeleton and nuclei using rhodamine phalloidin and DAPI (Beyotime Biotechnology, Shanghai, China). Cells were observed under laser-scanning confocal microscopy (LSCM; FV1200; Olympus, Tokyo, Japan). Next, cells were washed with PBS three times in the dark to remove FITC-labeled nano-TiO2, fixed with 4% paraformaldehyde at 37°C for 30 minutes, and permeabilized with 0.3% Triton X-100. After 10 minutes, cells were washed three times with PBS. To stain the cell skeleton, 100 nM rhodamine phalloidin was added and DAPI labeling performed for nuclear staining after 30 minutes. Internalization of nano-TiO2 in LCs was observed using LSCM.
3
Cellular Behavior on TiO2 Surfaces
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Morphology and morphometry of cells on TiO2 surfaces of all groups were examined after 24 hours following cell seeding to evaluate the cellular spreading and cytoskeletal arrangement of the cultured cells using confocal laser scanning microscopy. Cells were fixed in 10% formalin and confocal microscopic images of cells stained with 4′,6-diamidino-2-phenylindole (DAPI) to detect the nucleus (keygentec), antivinculin to detect vinculin protein within the cytoplasm (Proteintech), and rhodamine phalloidin for actin filaments (beyotime.inc).
4
Smp24 Peptide Characterization and Cytotoxicity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPMI-1640, fetal bovine serum (FBS), phosphate-buffered saline (PBS) and trypsin were all obtained from Gibco (New York, NY, USA). A549, H3122, PC-9, H460 and MRC-5 cells were purchased from the American Type Culture Collection (Manassas, WV, USA). Cells were grown in RPMI-1640 medium containing 1% penicillin–streptomycin and 10% FBS under the condition of 37 °C and 5% CO2. Lactate dehydrogenase (LDH) release assay kit, rhodamine–phalloidin and DAPI were obtained from Beyotime Institute of Biotechnology, Shanghai, China. Smp24 (IWSFLIKAATKLLPSLFGGGKKDS) was synthesized by GL Biochem Ltd. (Shanghai, China) and purified with an Inertsil ODS-SP (C-18) RP-HPLC column (Shimazu, Japan) to over 95% purity. The high-purity peptide was collected, lyophilized, and further identified by MALDI-TOF mass spectrometry (Figures S2 and S3 ). The theoretical mass of 2578.09 Da of the peptide coincides with the experimentally determined one, 2578.30 Da.
5
Immunofluorescence Staining of Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples were washed with PBS, fixed with 4 % paraformaldehyde for 30 min, according to experimental requirements, were incubated with anti-Pdpn (proteintech, 1:200), anti-Grem1 (CST, 1:50), anti-Cd200 (proteintech, 1:200), anti-Ctsk (Abcam, 1:200), anti-Ptgs2 (proteintech, 1:200), anti-vinculin (proteintech, 1:200) or anti-YAP (CST, 1:200) at 4 °C for 8–12 h. After that, samples incubated with goat anti-rabbit IgG (Servicebio, Alexa Fluor 488, 1:300), goat anti-rabbit IgG (Servicebio, Cy3, 1:300) or goat anti-mouse IgG (Servicebio, Alexa Fluor 488, 1:200) for 1 h at room temperature. Nuclear staining was conducted with DAPI (Sigma), and F-actin stained by rhodamine phalloidin (Beyotime). Tissue samples were fixed in 4 % paraformaldehyde. After decalcification, all samples were embedded in paraffin and sectioned at 6-μm slices. The tissue sections were immunofluorescently stained with primary antibodies targeting Sry (Santa Cruz, 1:200), CD11b (Servicebio, 1:500), TNF-α (Uscn Life Science Inc., 1:1000), IL-1β (Servicebio, 1:1200) and IL-10 (Servicebio, 1:1000), and the goat anti-rabbit IgG (Servicebio, Alexa Fluor 488, 1:400) and goat anti-rabbit IgG (Servicebio, Cy3, 1:500). DAPI was used to stain the nuclei. The images were observed by a fluorescence microscope (Olympus CKX53) or Nikon confocal microscope (Nikon).
6
Phagocytosis Assay with Fluorescent Particles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PHrodo™ Green E. coli BioParticles™ was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Stattic, Fludarabinum, C29, TAK-242 were bought from MCE (Monmouth Junction, NJ, United States). BAY 11-7085, SP610025, PD184352, LY294002, AZD0530 and Y-27632 were purchased from Selleckchem (Houston, TX, United States). Mouse IL-1β, IL-6, TNF-α Quantikine ELISA Kit, Cytochalasin D were bought from R&D Systems (Minneapolis, MN, United States). Mouse myeloperoxidase/MPO ELISA Kit was purchased from MultiScience (Lianke) Biotech Co., Ltd. (Hangzhou, China). p-STAT3 (Tyr705), p-STAT1 (Tyr701), p-p65(Ser536), p-Src (Tyr416), p-Lyn (Tyr507), p-SAPK/JNK (Thr183/Tyr185), p-p38 (Thr180/Tyr182), p-ERK1/2 (Thr202/Tyr204) were bought from Cell Signaling Technology (Beverly, MA, United States). STAT3, STAT1, p65, Src, Lyn, JNK, p38, ERK1/2 were purchased from wanleibio (Shenyang, China). β-actin was purchased from Bioworld Technology (Bloomington, MN, United States). FITC anti-mouse F4/80 antibody (clone BM8), APC anti-mouse CD64 (FcγRI) antibody (clone X54-5/7.1), PE anti-mouse CD16/32 antibody (clone 93), purified anti-mouse CD16/32 antibody (clone 93) and Ultra-LEAF™ Purified anti-mouse CD64 (FcγRI) antibody (Clone W18349F) were purchased from Biolegend (San Diego, CA, United States). Rhodamine phalloidin and DAPI were purchased from Beyotime (Nanjing, China).
7
Characterization of Cell-Membrane Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hPDLSCs were seeded onto the membranes at a density of 2 × 104 cells/well in a 24-well plate. Following a 3-day cultivation period, cells were treated with 4% paraformaldehyde for 10 min for fixation, followed by permeabilization using 0.1% Triton X-100 (Beyotime, China) for 5 min. Subsequent to blocking with 1% bovine serum albumin (BSA, Sigma Aldrich) for 30 min, the cells were stained with rhodamine phalloidin (Beyotime, China) for cytoskeleton and 4’, 6-Diamidino-2-phenylindole, dihydrochloride (DAPI, Beyotime, China) for the nucleus. The cellular structure and attachment to the membranes were examined using a confocal laser scanning microscope (CLSM, LSM 800, Carl Zeiss, Jena, Germany). The RAW 264.7 macrophages were cultured on the membranes with a density of 2 × 104 cells/well separately. Following a 3-day incubation, cells were fixed overnight with 2.5% glutaraldehyde. Subsequently, the fixed cells underwent dehydration using graded ethanol solutions (30%, 50%, 70%, 90%, and 100%) for 10 min respectively. Following dehydration, the specimens were subjected to freeze-drying and observed under SEM. In accordance with the protocol, the CCK8 assay was performed to assess the cell viability. Detailed methods were given in Supplementary Information.
8
Multinuclear Osteoclast Visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Multinuclear osteoclasts were fixed with 4% formaldehyde for 30 min and permeabilized with 0.2% Triton X-100 for 5 min. The cells were then blocked with 1% goat serum and 3% BSA and incubated with 2 U/ml rhodamine phalloidin (Beyotime, Shanghai, China) (1:1000 dilution) at room temperature for 30 min. Cell nuclei were stained with 1 μg/ml DAPI (Beyotime) for 1 min. Cells were visualized using a fluorescence microscope.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!