Mel270 cells were seeded in 8-chamber polystyrene-vessel tissue-culture glass slides (Corning, Kennebunk, ME, USA) at 3000 cells per chamber and allowed to attach for 24 hours. Cells were incubated with 300 pM AU-011 for 4 hours at 4°C or 37°C. Slides were washed with PBS and incubated with a cell surface marker (CD44-FITC; Thermofisher) at 4°C for 30 minutes. Then, the slides were washed 3 times with PBS and fixed with 1% formalin (J.T. Baker) for 10 minutes at room temperature. The cells were washed again with PBS and stained with DAPI (4',6-diamidino-2-phenylindole, Sigma, St. Louis, MO, USA) at 5 µg/mL for 5 minutes. Slides were again washed 3 times with PBS, after which coverslips were mounted on the glass slides using Mowiol mounting medium (Sigma) supplemented with 2.5% DABCO and sealed with nail polish. Slides were imaged on a Leica SP8 fluorescence microscope.
Sp8 fluorescence microscope
Manufactured by Leica
Sourced in United States
The Leica SP8 is a fluorescence microscope designed for high-performance imaging. It features a confocal scanning system that allows for detailed, high-resolution imaging of fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Mowiol mounting medium, by Merck Group (2 mentions)
Cd44 fitc, by Thermo Fisher Scientific (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Mouse anti eif4e, by BD (1 mentions)
Duolink detection kit, by Merck Group (1 mentions)
Rabbit anti eif4g, by Cell Signaling Technology (1 mentions)
Apollo reaction cocktail, by RiboBio (1 mentions)
8 chamber polystyrene vessel tissue culture treated glass slides, by Corning (1 mentions)
Dabco, by Merck Group (1 mentions)
Quickblock blocking buffer, by Beyotime (1 mentions)
Alexa fluor 488, by Cell Signaling Technology (1 mentions)
Af0131, by Affinity Biosciences (1 mentions)
Df6919, by Affinity Biosciences (1 mentions)
Fish assay kit, by Abnova (1 mentions)
Vimentin monoclonal antibody, by Abcam (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
12 protocols using sp8 fluorescence microscope
1
AU-011 Binding Assay in Mel270 Cells
2
Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% saponin in PBS for 5 min (optional). After three washed with PBS, the cells were blocked in 1% BSA, and incubated overnight at 4 °C with PBS containing 1% BSA and primary antibody (1:100). The cells were washed with PBS for three times followed by incubation with fluorescence‐labeled secondary antibody (1:300) for 1 h. Images were acquired with a Leica SP8 fluorescence microscope and quantified with the software ImageJ.
3
In situ Detection of eIF4E-eIF4G Interactions
4
Visualizing HSV-1 Uptake in BMDCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EdU‐labeled HSV‐1 were obtained by propagating HSV‐1 in Vero cells cultured in DMEM containing 10% FBS and 25 µm EdU (C00052, RIBOBIO). The cells and the supernatants were collected for three freeze‐thaw cycles to obtain EdU‐labeled HSV‐1. The BMDCs were incubated with EdU‐labeled HSV‐1 and cell Mask Green at 4 °C for 1 h (attachment) or at 37 °C for 15–30 min (penetration). Subsequently, the cells were then fixed in 4% paraformaldehyde and stained with Apollo reaction cocktail (C00031, RIBOBIO) at room temperature for 30 min. Images were captured on a Leica SP8 fluorescence microscope and processed with the ImageJ software for quantitative analysis.
5
Visualizing ZnPc-EV Cellular Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The location of the ZnPc-EVs inside cells was investigated by seeding 3000 MC38 cells in an 8-chamber polystyrene vessel tissue culture-treated glass slides (Corning) and allowing them to attach overnight. This was followed by incubation for 6 and 24 h with the ZnPc-EVs with a concentration calibrated at 4 µM ZnPc. After this time, the cells were washed 3 times in PBS and fixed in PBS containing 1% formalin (J.T. Baker) at 4 °C for 20 min. The cells were then washed three times in PBS and stained with 0.25 µM DAPI (Sigma), after which the coverslips were mounted on the glass slides using Mowiol mounting medium (Sigma-Aldrich) with 2.5% w/v DABCO (Merck) and sealed with nail polish. The slides were then imaged on a Leica SP8 fluorescence microscope.
6
Immunofluorescence Staining and Confocal Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 min, and then permeabilized in 0.2% Triton X-100 for 10 min. QuickBlock blocking buffer (P0252; Beyotime) was used to block nonspecific antigens for 2 h. Subsequently, the cells were incubated overnight with primary antibodies at 4°C. The cells were then incubated with secondary antibodies conjugated with Alexa Fluor 488 (Cell Signaling Technology [CST], 4412) or Alexa Fluor 594 (CST, 8889) for 1 h at 37°C. Finally, the cells were stained with DAPI (4′,6′-diamidino-2-phenylindole, catalog no. D9542; Sigma-Aldrich) for 10 min, washed three times with PBS, and analyzed by confocal microscopy with a Leica SP8 fluorescence microscope.
7
Adhesion Assay of B16-F10 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Adhesion of B16-F10 cells, incubated with platelets, to poly-L-lysine-coated glass coverslips was performed as described (Becker et al., 2017 (link)). Fixed cells were stained with anti-P-selectin + anti-goat Cy3 antibodies and phalloidin. Cells on the coverslip were analyzed with a Leica SP8 fluorescence microscope.
8
Intestinal Tissue Immunofluorescence Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intestinal samples were fixed overnight in 4% Carnoy’s fixative. Samples were then dehydrated in gradient ethanol (100%–95%−90%–75%−50%) and Xylene solution for at least 4 h. Then 4-µm paraffin sections were rehydrated, blocked with 3% BSA solution, and stained with antibodies overnight as follows: ZO-1 (Proteintech, 21773-AP, 1:2,000), E-cadherin (Affinity, AF0131, 1:200), and claudin-1 (Affinity, DF6919, 1:200). Tissue sections were then incubated with the appropriate fluorophore-conjugated secondary antibody. Before imaging, nuclei were counterstained with DAPI or Hoechst. Fluorescence analysis was performed on a Leica SP8 Fluorescence microscope.
9
Immunofluorescence Staining of Liver and Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining was performed, as previously described [35 (link)]. In brief, cryosections of the liver, and cultured cells, were fixed with 4% paraformaldehyde, permeated with PBS containing 0.2% Triton X-100 for 10 min. Specimens were then blocked with 5% goat serum before incubation with primary antibodies for 1 h at room temperature. After 3 washes with PBS, specimens were incubated for 30 min with Alexa Fluor–conjugated secondary antibodies, phalloidin and DAPI, washed, and then mounted with ProLong Diamond Antifade mounting medium for visualization under a Leica SP8 fluorescence microscope. Fluorescence intensity of the obtained images was quantified using ImageJ software.
10
Chromosome Enumeration by FISH
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FISH was performed according to protocol of the FISH assay kit (Abnova, Taiwan). Briefly, cells were placed on slides, and sequentially treated with methanol, acetic acid, 2× standard saline citrate, hot citric acid, and pepsin. Centromere-specific α-satellite (CEN) probes for chromosomes 1 (Texas Red), and 5 (Cy5) were added to each slide, the DNA and probe were codenatured and hybridized, and the slides were washed and counterstained. Cells were then viewed with a Leica SP8 fluorescence microscope. For each chromosome, >30 nuclei were analyzed.
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!