Fibroblasts from neonatal rats were seeded in a 48-well plate at 2 × 105 cells/well. Fibroblasts were treated the day before the analysis with 10 ng/mL of TGF-β to stimulate differentiation into myofibroblasts. Cells were then treated with 30 μg/mL of EVs or transfected with 20 nM miR-4732-3p using lipofectamine as indicated above. A straight line in the monolayer of fibroblast was created using a 20-μL pipette tip. Images were taken 24 h after the addition of treatments using a Leica DM600 inverted microscope at 4 × magnification. The area of the scratch wound was then measured using ImageJ.
Dm600 inverted microscope
Manufactured by Leica
The Leica DM600 is an inverted microscope designed for various laboratory applications. It features a stable and ergonomic design, providing a platform for detailed observation and analysis of samples. The DM600 offers high-quality optics and illumination to enable clear, high-contrast imaging.
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2 protocols using dm600 inverted microscope
1
Myofibroblast Differentiation and Migration
2
Immunofluorescence Staining of RPE-1 BRAF V600E Cells
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RPE-1 BRAF V600E cells were grown directly on collagen IV (Sigma) -coated coverslips, fixed in 3.7% formalin, permeabilized using 0.1% Triton X-100, and treated with 0.1% SDS. They were blocked in 1% BSA and then incubated with primary antibody diluted in blocking solution in a humidity chamber at 4°C overnight, washed with 1X PBS, and incubated with secondary antibody. Cells were mounted using mounting media containing DAPI (Vector Laboratories). All secondary antibodies were Alexa Fluor conjugates (488, 555, and 647) (Thermofisher) used at a 1:500 dilution. All images were acquired on a Leica DM 600 inverted microscope at 100X magnification. Z stack images were taken at 0.20µM each.
For melanocyte binucleate counts and immunofluorescence staining, zebrafish adult dorsal scales were fixed using 4% paraformaldehyde for two hours, washed with PBST (PBS+ 0.1% Triton X) and water then blocked in 1%BSA/PBS for 30 minutes prior to primary antibody incubation. Affinity-purified anti-Mitfa antibodies were used at a 1:100 dilution for staining. All images were acquired on a Nikon Eclipse Ti A1R-A1 confocal microscope. Additional information is provided in Extended Experimental Procedures.
For melanocyte binucleate counts and immunofluorescence staining, zebrafish adult dorsal scales were fixed using 4% paraformaldehyde for two hours, washed with PBST (PBS+ 0.1% Triton X) and water then blocked in 1%BSA/PBS for 30 minutes prior to primary antibody incubation. Affinity-purified anti-Mitfa antibodies were used at a 1:100 dilution for staining. All images were acquired on a Nikon Eclipse Ti A1R-A1 confocal microscope. Additional information is provided in Extended Experimental Procedures.
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