Pre-coated growth-factor reduced Matrigel cell culture inserts (24-well, pore size 8 μm; BD Biosciences, San Jose, CA, USA) were seeded with serum-deprived 5 x 105 PC3 or 22Rv1 vector-only cells (22Rv1-VO) or 22Rv1 EphB4 over-expressing cells (22Rv1-B4) and transfected with 100 nM ITGB8 siRNA or non-silencing AllStars siRNA (Qiagen) in 0.1% FCS-containing medium. Medium containing 10% FCS was used as chemo-attractant. Cells were incubated for 22 h and cells that had not invaded were removed from the upper chamber using a cotton swab. Membranes were excised and mounted on glass slides with ProLong Gold Antifade containing 4, 6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Life Technologies) for visualization of the nuclei of cells that had invaded through the Matrigel to the underside of the membrane. Nuclei were counted in five random fields at 20 X magnification using an Olympus epifluorescent microscope.
Epifluorescent microscope
Manufactured by Olympus
Sourced in Japan, United States
The Epifluorescent microscope is an optical microscope designed for observing and analyzing fluorescently labeled samples. It uses a high-intensity light source, such as a mercury or xenon lamp, to excite fluorescent dyes within the specimen, allowing for the visualization of specific structures or molecules. The epifluorescent microscope is a versatile tool used in various fields of research, including cell biology, molecular biology, and material science.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 546 goat anti rabbit igg antibody, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Rabbit anti oct4, by Cell Signaling Technology (2 mentions)
Dapi nuclear dye, by Merck Group (2 mentions)
Mouse anti sox2, by Cell Signaling Technology (2 mentions)
Mouse anti nanog, by Cell Signaling Technology (2 mentions)
Rabbit anti ki67, by Abcam (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Protein block solution, by Agilent Technologies (2 mentions)
Saponin, by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Allstars sirna, by Qiagen (1 mentions)
Image pro plus 5, by Media Cybernetics (1 mentions)
Tgf β1, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33342 stain, by Thermo Fisher Scientific (1 mentions)
Dako fluorescent mounting medium, by Agilent Technologies (1 mentions)
Ab8295, by Abcam (1 mentions)
Cryostat, by Leica (1 mentions)
Ab184787, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
29 protocols using epifluorescent microscope
1
Invasion assay for prostate cancer cells
2
Histological and Immunohistochemical Analysis of LV Myocardium
3
Quantifying Apoptotic Nuclei via Hoechst Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment with epigenetic modulators, cells were stained with Hoechst 33342 stain (1 mg/mL, Invitrogen) followed by incubation for 10 min at 37°C. Images were taken under UV filter using Epi-fluorescent Microscope (Olympus IX71) at 400 X magnification with an excitation wavelength of 355 to 366 nm and an emission wavelength of 465 to 480 nm. Condensed nuclei were counted against total number of nuclei in the field, and the percentage of apoptotic nuclei were calculated and plotted graphically.
4
Flii and Collagen Expression in Digit Wound Healing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical experiments were undertaken on all digits collected from Balb/c WT and FliiTg/Tg mice at days 0, 3, 7, 14, 21 and 28 post-surgery. Following antigen retrieval, 3% normal goat serum diluted in PBS was used for blocking for 30 min. Primary antibodies were used at 2 μg/ml and included mouse α-Flii (Santa Cruz sc-21716), rabbit α-Collagen I (Rockland 600-401-103), rabbit α-Collagen III (Rockland 600-403-105), mouse α-TGFβ1 (Santa Cruz sc-52893) and rabbit α-TGFβ3 (Santa Cruz sc-83). Species-specific Alexa Fluor 488, 568 or 633-conjugated secondary antibodies (1:400, Invitrogen, Carlsbad, USA) were diluted in PBS and applied for detection. Nuclear counterstain 4,6-diamidino-2-phenyindole (DAPI) was applied last. The slides were mounted in Dako Fluorescent Mounting Medium (DAKO Corporation, Sydney, Australia) and viewed using an Olympus Epifluorescent microscope.
5
Histological and Immunofluorescence Analysis of Murine Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice tissues were harvested and fixed using a neutral buffered 10% formalin solution (NBF, Sigma-Aldrich, St. Louis, MO, USA) overnight. Afterward, the tissues were dehydrated in a 30% sucrose solution at 4°C for at least one night. Subsequently, the tissues were cryopreserved in optimal cutting temperature (OCT) compound and cryosectioned (CryoStats, Leica). Pathological assessments were carried out using H&E and Masson’s trichrome staining in accordance with the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA).
For immunofluorescence staining, cryosections of the tissues were first permeabilized and blocked using a protein block solution (Dako, Carpinteria, CA, USA) containing 0.1% saponin (Sigma-Aldrich, St. Louis, MO, USA). The primary antibodies were then added and incubated overnight at 4°C, including rabbit anti-Caspase 3 (1:100; ab184787, Abcam, Cambridge, UK), rabbit anti-Ki67 (1:100; ab15580, Abcam), anti-CD31 antibody (1:100; EPR17259, Abcam), and mouse Anti-Cardiac Troponin T antibody (1:100; ab8295, Abcam) to target proteins of interest. Fluorophore-conjugated secondary antibodies were then added for fluorescent imaging. All the tissue slides were mounted by ProLong gold antifade mountant with DAPI (Thermo Fisher, US) before being imaged using an epifluorescent microscope from Olympus.
For immunofluorescence staining, cryosections of the tissues were first permeabilized and blocked using a protein block solution (Dako, Carpinteria, CA, USA) containing 0.1% saponin (Sigma-Aldrich, St. Louis, MO, USA). The primary antibodies were then added and incubated overnight at 4°C, including rabbit anti-Caspase 3 (1:100; ab184787, Abcam, Cambridge, UK), rabbit anti-Ki67 (1:100; ab15580, Abcam), anti-CD31 antibody (1:100; EPR17259, Abcam), and mouse Anti-Cardiac Troponin T antibody (1:100; ab8295, Abcam) to target proteins of interest. Fluorophore-conjugated secondary antibodies were then added for fluorescent imaging. All the tissue slides were mounted by ProLong gold antifade mountant with DAPI (Thermo Fisher, US) before being imaged using an epifluorescent microscope from Olympus.
6
Quantifying Cardiac Fibrosis and Myocyte Size
7
Chlortetracycline Staining of Sperm Capacitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chlortetracycline (CTC) staining was carried out according to the previously described protocol [34 (link)]. Chlortetracycline solution (750 mmol/L CTC, 5 mmol/L cysteine in 130 mmol/L NaCl and 20 mmol/L Tris-HCl, pH 7.4) was freshly prepared, and pH was adjusted to 7.8. The control and treated groups were taken at each hourly interval during 4 h incubation period, mixed with an equal volume of CTC solution and after a few seconds, 1 mL of 4% paraformaldehyde was added. A thin smear was made on a glass slide, and a drop of DABCO was added before applying the cover slip to retard the fading of the CTC fluorescence. Chlortetracycline fluorescence was observed using an epifluorescent microscope (Olympus, USA). The assessed sperms were classified into either uniform bright fluorescence over the whole head (uncapacitated sperm, pattern F), a fluorescence-free band in the post-acrosomal region (capacitated sperm, pattern B) and dull fluorescence over the entire head (acrosome- reacted sperm, pattern AR).
8
Immunofluorescent Imaging of Mitochondria in Melanoma Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formaldehyde-fixed melanoma cells were stained with mouse anti-Tom20 antibody (F-10, sc-17764, Santa Cruz biotechnology, Heidelberg, Germany) and Alexa Fluor 488 goat anti-mouse antibody (R37120, Thermo Fischer Scientific), counterstained with DAPI (Sigma), and imaged using an epi-fluorescent microscope (Olympus, Japan) with a 60 × lens. For staining of the active mitochondria, the melanoma cells were stained with 100 nM MitoTracker Red CMXRos (#9082, Cell Signaling Technology, Frankfurt, Germany) for 20 minutes at 37°C, fixed with ice-cold methanol, and imaged using an epi-fluorescent microscope.
9
3D Collagen Gel Contraction Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary tenocytes and fibroblasts isolated from Flii+/−, WT, and FliiTg/Tg mice tendons or skin, respectively, were trypsinized in 10 × trypsin/EDTA (Sigma-Aldrich, Sydney, Australia) for 5 min at 37 °C and quenched using DMEM + 20% FBS. The cells were spun for 5 min at 1200 rpm and resuspended in ice-cold DMEM at 1 × 106 cells/mL. 3D collagen gels were prepared by mixing 8 parts of chilled collagen solution (2 mg/mL) with 1 part 10 × DMEM containing 10% FBS as previously described [16 (link)]. The pH was adjusted to 7.4 using 0.1 M NaOH. The tenocytes and fibroblasts were added to the collagen gel mixture at 1 × 105 cells/mL. Five hundred microliters of this mixture was added per well to a flat-bottomed 48-well plate and allowed to set for 120 min at 37 °C 5% CO2. Following this, the gel was carefully dislodged using a 200-μL pipette and the gels floated by the addition of 1 mL DMEM (+ 20% FCS, Pen/Strep, and Fungizone). Images were taken at 24, 48, and 72 h post media addition using Olympus Epifluorescent microscope and the images analyzed for degree of contraction using Image Pro-Plus 7.1 as previously described [16 (link)].
10
GFAP Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultures were fixed for 25 min in cold 4% paraformaldehyde (PFA) in PBS at room temperature. After being washed three times (5 min each time) with PBS, fixed samples were permeabilized in 0.3% Triton X‐100 solution for 20 min and blocked with 5% normal goat serum for 1 hr at room temperature. Primary antibodies and the mouse antiglial fibrillary acidic protein antibody (anti‐GFAP; 1:1,000; Cell Signaling Technology, USA) in the blocking solution were applied overnight at 4°C. After rinsing, samples were incubated with secondary antibody conjugates for mouse IgG (1:200, Cell Signaling Technology) in blocking solution for 2 hr at room temperature. After completion of the secondary antibody incubation, the nuclear stain 4,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI) was added for 5 min. Excess secondary antibody and nuclear counterstain were removed by washing in PBS. Fluorescent‐labeled samples were coverslipped with fluorescent mounting medium. Images were captured using an epifluorescent microscope (Olympus, Japan) equipped with the digital microscope system (Olympus, Japan).
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!