Cells of SCC25 and RAW 264.7 were placed into the transwell inserts, treated as above. To obtain osteoclasts, RAW 264.7 were supplemented with 50 ng/ml of recombinant mouse RANKL (Pepro Tech). Medium change and new cytokine were added every 2 days, while osteoclasts appeared after 4 days of treatment. These osteoclasts were subsequently fixed in 10% formalin. TRAP staining and IF of F-actin staining were used to characterize osteoclasts. TRAP positive cells of three or more nuclei were considered to be multinucleated osteoclasts. Rhodamine-conjugated phalloidin (Life Technologies) were used to stain F-actin. DAPI staining (Boster) was used to visualize the nuclei. Four fields were randomly selected and counted for osteoclast and F-actin numbers.
Dapi staining
Manufactured by Boster Bio
Sourced in China
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) is a fluorescent dye that binds strongly to adenine-thymine (A-T) rich regions in DNA. It is commonly used in fluorescence microscopy as a nuclear counterstain to visualize cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 900 confocal microscope, by Zeiss (3 mentions)
Recombinant mouse rankl, by Thermo Fisher Scientific (1 mentions)
Rhodamine conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Spider βgal, by Dojindo Laboratories (1 mentions)
Ar1177, by Boster Bio (1 mentions)
Fluorescein isothiocyanate (fitc), by Boster Bio (1 mentions)
Inverted microscope, by Nikon (1 mentions)
4 protocols using dapi staining
1
Osteoclast Formation Assay
2
SPIDER-β-gal Assay for Cell Imaging
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After fixing the cells for 15 min with 4% paraformaldehyde, they were treated for 15 min at 37 °C with 1 μmol/L SPIDER-β-gal (SG02, Dojindo, Kumamoto, Japan). DAPI staining (AR1177, Boster Bio, Shanghai, China) was applied to the nuclei for 10 min. Following the wash with PBS, cells were observed using the LSM900 confocal microscope (Zeiss, Oberkochen, Germany).
3
E-cadherin Expression Analysis by IF
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To further confirm the switch of E-cad protein, immunofluorescence (IF) was utilized. Cells of SCC25 and RAW 264.7 were placed into transwell inserts and treated as above. On each time point, both cells of SCC25 and RAW 264.7 were fixed with 75% ethanol for 10 min, and blocked in 5% bovine serum albumin for 30 min. Primary antibody of E-cad (1:50) were incubated at 4°C overnight, followed by detection with fluorescence-conjugated secondary antibody (FITC, 1:200, Boster) at room temperature for 2 h. DAPI staining (Boster) was used to visualize the nuclei. Images were acquired and photographed by a Nikon inverted microscope. Normal serum replaced primary antibodies as negative controls.
4
Visualizing Actin Filaments in NP-MSCs
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Rhodamine-labelled phalloidin (Cytoskeleton, Denver, CO) was used to stain the actin filaments in NP-MSCs. Briefly, after being treated, the cells were fixed in 4% formaldehyde for 10 min at room temperature and permeabilized with 0.3% Triton X-100 in PBS for 15 min. The cells were incubated with rhodamine-phalloidin (100 nM) in the dark for 30 min. Then, the cells were washed two times and incubated with DAPI staining (Boster, China) for 5 min in the dark. After being washed two times with PBS, the cells were observed and photographed by using a confocal microscopy (Nikon A1, Japan).
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