Cells were fixed, permeabilized with 0.5% Triton X-100 (Beyotime, Shanghai, China), and blocked with 5% BSA. Primary antibodies specific to β-catenin (dilution 1:200, EM0306, Huabio, Hangzhou, China), LGR6 (dilution 1:200, ab126747, Abcam, Shanghai, China) and HnRNPK (dilution 1:200, SC60-03, Huabio, Hangzhou, China) were then added and incubated overnight at 4 °C. Secondary antibodies labeled with Cy3 (A0521, Beyotime, Shanghai, China) or Alexa Fluor 555 (ab150074, Abcam, Shanghai, China) were used to incubate the samples for 2 h. DAPI (S2110, Solarbio, Beijing, China) and phalloidine (6 μmol/L, Invitrogen, Carlsbad, CA, USA) were applied to stain the nuclei and cytoskeleton. Finally, cell samples were observed with CLSM.
Phalloidine
Manufactured by Thermo Fisher Scientific
Sourced in United States
Phalloidine is a fluorescent dye used in biological research to specifically label and visualize actin filaments in cells. It binds tightly to F-actin, allowing for the detection and localization of the actin cytoskeleton.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Beyotime (3 mentions)
D9542, by Merck Group (2 mentions)
Ab126747, by Abcam (1 mentions)
Dapi s2110, by Solarbio (1 mentions)
Ab150074, by Abcam (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Smad3, by Abcam (1 mentions)
Anti cx43, by Abcam (1 mentions)
Repsox, by Abcam (1 mentions)
Pannexin 1, by Abcam (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Ab150075, by Abcam (1 mentions)
Tcs sp5, by Leica (1 mentions)
Cell death detection kit, by Roche (1 mentions)
Ab150113, by Abcam (1 mentions)
Ab150079, by Abcam (1 mentions)
Rna isolation kit, by BioTeke (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Live dead reagent, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
7 protocols using phalloidine
1
Immunofluorescence Analysis of β-catenin, LGR6, and hnRNPK
2
Immunofluorescence Staining of Cx43, Pannexin1, and Smad3
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Immunofluorescence staining were performed as previously described36 (link). Briefly, Cells were cultured in Petri dishes specified for confocal laser microscopy for 12 h. Then TGF-β1 (5 ng/ml, p04202, R&D Systems, USA) and Repsox (50 μM, ab142139, Abcam, Cambridge, UK) were added into the culture media respectively and continued to incubate for 24 h. Then the cells were fixed in 4% paraformaldehyde for 20 min, and rinsed with PBS three times, permeabilized with 0.5% Triton X-100 (Beyotime, Shanghai, China) for 15 min, and blocked with 5% BSA for 1 h. The samples were then incubated with the Anti-Cx43 (1:200; Abcam, Cambridge, UK), pannexin1 (1:200; Abcam, Cambridge, UK), Smad3 (1:200; Abcam, Cambridge, UK) rabbit monoclonal antibodies overnight at 4 °C. The secondary antibody was Alexa Fluor® 647 (10 μg/ml, Alexa Fluor ®647, Life Technology, Grand Island, NY, USA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; D9542, Sigma, USA) and phalloidine (6 μM, Invitrogen, CA) was used to label the cytoskeleton.
3
Chondrocyte Cx43 and p-AKT Regulation
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Chondrocytes were seeded and cultured in observation dishes specified for confocal laser microscopy (CLSM) for 12 hours. The effect of CTGF and LY294002 on the expression of Cx43 and p‐AKT was detected in chondrocytes by CLSM. Chondrocytes were firstly treated with 50 ng/mL CTGF for 2 and 12 hours with presence and absence of 10 μmol/L LY294002 to detect the expression of p‐AKT and Cx43, respectively. The culture medium was discarded and cells were rinsed with PBS three times and then fixed with 4% cold paraformaldehyde solution, permeabilized with 0.5% Triton X‐100 (Beyotime, China, Shanghai) for 10 minutes, and blocked with 5% BSA for 1 hour. After being washed with PBS three times, primary antibodies against Cx43 (1:300, Abcam, Cambridge, UK) and p‐AKT (1:200, 340705, zen‐bio, China) were added into the dishes and incubated at 4°C overnight, respectively. A secondary antibody conjugated to AlexaFluor647 (ab150075, Abcam) was used to incubate the samples for 2 hours. DAPI (D9542, Sigma, USA) and phalloidine (6 μmol/L, Invitrogen, CA) were applied to stain the nuclei and cytoskeleton. The cells then were observed with CLSM. All experiments were repeated at least three times.
4
Kidney Cell Death Quantification
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Kidney was fixed in 4% PFA solution for 48 h for preparation of paraffin section (4 μm). Sections were incubated overnight at 4°C with phalloidine (Invitrogen, California, USA) for F-actin staining and then subjected to TUNEL staining using a cell death detection kit (Roche, Basel, Switzerland), according to the manufacture's instruction, and the nuclei were labeled with hoechst 33342. Five fields were selected from renal cortex for each section at × 40 magnification of objective, and observed with a Laser Scanning Confocal Microscope (TCS SP5, Leica, Mannheim, Germany). The numbers of the TUNEL-positive cells in the five fields were counted, and the average was calculated.
5
hPDLSCs Immunofluorescence and CLSM Protocol
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The detailed hPDLSCs treatment protocols were in accordance with ALP staining experiments above. RNA was collected in accordance with the instructions of the RNA Isolation Kit (Bioteke, Beijing, China.), and then 1 µg RNA was transformed into cDNA with reverse transcriptase (Thermo Fisher Scienti c, MA, USA) and subjected to quantitative RT-PCR reaction (Bio-Rad, CA, USA). The relative expression of targeted genes (Table. S1) was calculated applying the ΔΔCt method as previously described. [30] (link) 2.9 Immuno uorescence and Confocal Laser Scanning Microscope (CLSM)
The detailed immuno uorescence procedure was described formerly [25] . The hPDLSCs were xed in 4% PFA for 15 min. Subsequently, hPDLSCs or tissue sections were permeated with 0.25% Triton X-100 for 10 min. The hPDLSCs or tissue sections were reacted with anti-Cx43 and anti-β-catenin antibodies overnight at 4℃ after being sealed with 5% BSA for 1 h. Anti-rabbit antibody AlexaFluor647 (ab150079, Abcam, UK) or anti-mouse antibody AlexaFluor488 (ab150113, Abcam, UK) was incubated at room temperature for 2h. DAPI (D9542, Sigma, America) staining was performed for periodontal ligament cells nuclei. The cytoskeletons were dyed with phalloidine (6µmol/L, Invitrogen, CA). Fluorescence staining images were visualized by CLSM.
The detailed immuno uorescence procedure was described formerly [25] . The hPDLSCs were xed in 4% PFA for 15 min. Subsequently, hPDLSCs or tissue sections were permeated with 0.25% Triton X-100 for 10 min. The hPDLSCs or tissue sections were reacted with anti-Cx43 and anti-β-catenin antibodies overnight at 4℃ after being sealed with 5% BSA for 1 h. Anti-rabbit antibody AlexaFluor647 (ab150079, Abcam, UK) or anti-mouse antibody AlexaFluor488 (ab150113, Abcam, UK) was incubated at room temperature for 2h. DAPI (D9542, Sigma, America) staining was performed for periodontal ligament cells nuclei. The cytoskeletons were dyed with phalloidine (6µmol/L, Invitrogen, CA). Fluorescence staining images were visualized by CLSM.
6
Fluorescent Cytoskeleton and Nucleus Staining
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Cells (2 × 104) were planted on the carry sheet glass (48‐well size) and allowed to grow to 60% density. Then cells were fixed by 4% paraformaldehyde for 30 minutes. Phalloidine (1:500 diluted, A12379, Thermo Fisher, USA) was used to stain the fixed cells for 1 hour in absence of light. DAPI (C0065, Solarbio, China) was used to stain the nucleus.
7
Rabbit BMSC Isolation and Characterization
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Primary rabbit BMSCs (rBMSCs) were harvested from femur bone marrow of 3 weeks old rabbit. Cell culture medium (CM) and fetal bovine serum were purchased from Gibico (USA). Cells at passage 3–5 were used in the following experiments. Recombinant IGF-1 protein was purchased from Peprotech (100–11, Peprotech, USA). Cell Counting Kit 8 (CCK-8) was purchased from Dojindo (Shanghai, China). Live/dead reagent, Phalloidine and Second antibody Alexa Flour 555 were purchased from Thermofisher. Annexin V-FITC apoptosis assay kit was purchased from Absin (Shanghai, China). Type II collagen antibody was purchased from Norvas (NB600-844, USA).
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