For the in situ determination of the chemical composition of intracellular structures, a confocal Raman microscope (alpha300 RSA; WITec, Germany) was used as previously described (56 (link)– (link)60 (link)). To immobilize the fast-moving flagellates on the quartz slide, 5 μL of the cell pellet was mixed with 5 μL of 1% (wt/vol) solution of low-melting-point agarose (catalog number 6351.5; Carl Roth, Germany), immediately spread as a single-cell layer between a quartz slide and coverslip, and sealed with CoverGrip sealant (Biotium, USA). Two-dimensional Raman maps were obtained with laser excitation at 532 nm (20 mW power at the focal plane) and oil-immersion objective UPlanFLN 100×, numerical aperture (NA) 1.30, or water-immersion objective UPlanSApo 60×, NA 1.20 (Olympus, Japan). A scanning step size of 200 nm in both directions and an integration time of 100 ms per voxel were used. A minimum of 30 cells were measured for each strain. Raman chemical maps were constructed by multivariate decomposition of the baseline-corrected spectra into the spectra of pure chemical components by using Project Plus 5.1 software (WITec, Germany).
Covergrip sealant
Manufactured by Biotium
Sourced in United States
CoverGrip Sealant is a clear liquid sealant designed to secure microscope coverslips to glass slides. It provides a durable and long-lasting bond between the coverslip and slide, helping to prevent sample loss or displacement during handling and storage.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Ckx41 fluorescence microscope, by Olympus (2 mentions)
Uplansapo 60, by Olympus (1 mentions)
Alpha300 rsa, by WITec (1 mentions)
Project plus 5, by WITec (1 mentions)
Eclipse 90i, by Nikon (1 mentions)
Ds ri1, by Nikon (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Prolong glass, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Low gelling agarose, by Merck Group (1 mentions)
8 protocols using covergrip sealant
1
Intracellular Chemical Composition Analysis by Raman Microscopy
2
Immunofluorescence Analysis of CB1 and CB2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RCC cells were cultured in 4-chamber slides as described above. At approximately 80% confluence, the monolayer cell culture was rinsed briefly in PBS. Next, the cells were covered in 4% paraformaldehyde (PFA) for 10 min at room temperature. The PFA was removed by washing with PBS (3 times) and goat serum (10%) in PBS was used for blocking for 1 h at room temperature. The cells were incubated separately with diluted primary antibodies against CB1 (1:1000) and CB2 (1:1000) at 4 °C for 4 h. The cells were washed three times with PBS and then incubated with Alexa Fluor® 546 secondary goat anti-rabbit antibody (1:400) for 2 h at room temperature away from light. Again, the cells were rinsed 3 times with PBS, followed by incubation with DAPI (1:5000) (ThermoFisher Scientific, Massachusetts, USA) for 10 min. For the control, the cells were incubated only with secondary antibody. The slides were washed with PBS and covered with coverslips using CoverGrip Sealant (Biotium, California, USA), and images were captured using an Olympus CKX41 fluorescence microscope.
3
RCC Sphere Immunophenotyping Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RCC cells were counted and seeded at density of 100 cells/well in ultra-low attachment 24 wells plates (TC plate, suspension, F, Sarstetd, Numbrecht, Germany) supplemented with sphere promoting media as described previously20 (link),77 (link). Later culture media was removed, and tumor spheres were rinsed briefly in PBS (3 times). 10% goat serum in PBS was used for blocking at room temperature (1 hr). Tumor spheres were incubated separately with diluted primary antibodies against CD105 (1:1000), CD133 (1:500), CD44 (1:1000) and CXCR4 (1:1000) at 4 °C for 4 h; washed three times with PBS and incubated with Alexa Fluor 488 secondary goat anti-mouse antibody (1:400) for 1 h at room temperature. The spheres were rinsed 3 times with PBS, followed by incubation with DAPI (1:5000; ThermoFisher Scientific, Massachusetts, USA) for 10 min. As control the spheres were incubated only with secondary antibody. The slides were washed with PBS and covered with coverslips using CoverGrip Sealant (Biotium, California, USA), and images were captured using an Olympus CKX41 fluorescence microscope.
4
Deoxygenation and Polymerization of Sickle Hemoglobin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sickle hemoglobin (H0392; Sigma-Aldrich, St. Louis, MO) samples were suspended in 0.1 M potassium phosphate buffer (pH 7.0). Before imaging, HbS samples were deoxygenated on ice under a vacuum for 1 hour, after which samples were then brought to the final imaging concentration by further dilution in potassium phosphate buffer with sodium metabisulfite (final concentration of 50 mM) as an oxygen scavenger. Samples were placed on glass slides, covered with an acid-cleaned coverslip (6 (link)), and then sealed with CoverGrip sealant (Biotium, Fremont, CA). All samples were prepared at 4°C. Polymerization was initialized by temperature jump upon placing the slide on the microscope stage, where temperature was maintained at 25° or 37°C by use of an airstream incubator (Nevtek, Burnsville, VA) in combination with an objective heater (OkoLab SRL, Pozzuoli, Italy). Three independent sample preparations were performed for each temperature.
5
Immunofluorescence Analysis of ApoD in Astrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured IMA2.1 astrocytes attached to poly-L-lysine (Sigma)-treated coverslips were fixed with 4% formaldehyde, washed in phosphate buffered saline (PBS), blocked and permeabilized with Tween-20 (0.1%) and 1% non-immune calf serum. We used a goat polyclonal anti-mouse ApoD (SC Biotechnology) as primary antibody, and Alexa 488-conjugated donkey anti-goat IgG serum (Jackson Immunoresearch) as secondary antibody. Coverslips were mounted with EverBrite™-DAPI Mounting medium, and sealed with CoverGrip™ sealant (Biotium). Cells were visualized and photographed with an Eclipse 90i (Nikon) fluorescence microscope equipped with a DS-Ri1 (Nikon) digital camera, and images were processed and analysed with the Fiji Program.
6
Fluorescence Imaging of Brain Slices
7
Immunofluorescence Staining and Confocal Imaging
8
Raman Microscopy of Synchronized Algae
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To study the content and intracellular distribution of storage biomolecules during the cell cycle using Raman microscopy, fresh living cells were needed because of problematic photobleaching of autofluorescence in chemically fixed or frozen cells. Samples from a synchronized culture of D. quadricauda were taken every 30–60 min, as permitted by the duration of the Raman scanning, from the start of the light phase (T = 0:00 h), considered as the beginning of the cell cycle, up to the end of the cycle (T = 24:00 h); the first sample was taken 15 min after the start of the light phase.
For Raman measurements, 0.5 mL of the cell suspension was centrifuged (2000× g for ca. 15 s), the supernatant was discarded and a part of the pellet was mixed with ca. 20 µL of low-gelling agarose (Sigma Aldrich, 2% solution, T = 39 °C). A few µL of the agar mixture were placed between a quartz slide and a quartz coverslip, and these were sealed with Covergrip sealant (Biotium, Hayward, CA, USA). Sample preparation took about 13 min from taking the sample to the start of the Raman measurement.
For Raman measurements, 0.5 mL of the cell suspension was centrifuged (2000× g for ca. 15 s), the supernatant was discarded and a part of the pellet was mixed with ca. 20 µL of low-gelling agarose (Sigma Aldrich, 2% solution, T = 39 °C). A few µL of the agar mixture were placed between a quartz slide and a quartz coverslip, and these were sealed with Covergrip sealant (Biotium, Hayward, CA, USA). Sample preparation took about 13 min from taking the sample to the start of the Raman measurement.
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!