Cells were seeded on glass coverslips in 12-well plates (per well 2 × 105 081R cells, 1 × 105 289B cells), to allow imaging and exposed to 1Gy for 72 h. Cells were washed twice with PBS, fixed using 4% formaldehyde in PBS for 10 min, washed again and permeabilized using 1% Triton X-100 in PBS. Blocking was done using 5% goat serum (NGS)/0.1% Triton X100/PBS for 30 min. Antibodies were incubated overnight at 4°C in blocking buffer at the following dilutions: anti-ESRRA at 1:100 dilution (Merck), anti-Beta-I tubulin at 1:100 (Sigma). After washing, secondary antibodies were added: Alexa 568 anti-mouse IgG1 and Alexa 488 anti-rabbit IgG (H+L), both 1:100 (Invitrogen). Hoechst was added 1:1000. After further washing, cells were mounted in Prolong Gold and imaged on an SP-8 confocal microscope (Leica).
Prolong gold
Manufactured by Leica
Prolong Gold is a mounting medium designed for microscopy applications. It is a water-based formulation that provides enhanced signal preservation and reduced photobleaching of fluorescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Alexa568 anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Softworx, by GE Healthcare (1 mentions)
Volocity software package, by PerkinElmer (1 mentions)
Softworx software, by GE Healthcare (1 mentions)
Tcs sp2, by Leica (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Alexa fluor 555 phalloidin, by Thermo Fisher Scientific (1 mentions)
3 protocols using prolong gold
1
Immunofluorescence Imaging of 081R and 289B Cells
2
Wide-field and Superresolution Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Imaging was performed with an image restoration system (DeltaVision, GE Healthcare) using a wide-field microscope (IX71; Olympus) and a 100× 1.4-NA objective at room temperature. Samples were mounted in ProlongGold (Life Technologies), and a cooled charge-coupled device camera (CoolSNAP HQ2; Photometrics) was used. Images were subsequently processed by iterative constrained deconvolution (softWoRx; GE Healthcare) and corrected for chromatic aberrations. The Volocity software package was used to produce extended-view images (PerkinElmer). Neural tube images were acquired on a Leica TCS Confocal SP5-II confocal microscope with a 20× 0.7-NA water objective, and samples were mounted in ProlongGold. For superresolution microscopy, samples were mounted in ProlongDiamond (Life Technologies). Superresolution imaging was performed at room temperature on a DeltaVision OMX Blaze 3D-SIM superresolution microscope (Applied Precision) with a 100× 1.4-NA UPLSAPO oil objective (Olympus) and three-evolve electron-multiplying CCD cameras (Photometrics) for sequential acquisition. Images were subsequently processed with softWoRx software (GE Healthcare) and Imaris (Bitplane).
3
Visualizing F-Actin Dynamics in Cell Monolayers and Rounded Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For visualisation of F-actin structure in cell monolayer, cells cultured on cover slips were transfected with LifeAct-GFP or actin-GFP, fixed in 4% paraformaldehyde (PFA) for 10 min, permeabilised for 5 min in 0.5% Triton X-100/phosphate buffered saline (PBS) and stained with Alexa Fluor 555-phalloidin (1:40; Invitrogen) at 25 μl/ml in PBS+0.1% bovine serum albumin (BSA; Sigma-Aldrich) for 20 min. Coverslips with cells were then washed in PBS and mounted with ProLong Gold (Invitrogen).
For visualisation of F-actin structure in rounded cells, the following procedure was performed. Transfected cells with LifeAct-GFP or actin-GFP were detached using trypsin, suspended in imaging media and fixed in 4% PFA for 10 min, followed by permeabilisation in 0.5% Triton X-100/PBS (Sigma-Aldrich) for 5 min prior to staining with Alexa Fluor 555-phalloidin (1:40; Invitrogen) in PBS+0.1% BSA for 20 min. Cells were then washed in PBS and suspended in distilled water. A drop of stained cells in suspension was placed on a coverslip and allowed to dry. Coverslips with cells were mounted using ProLong Gold and imaged using a laser scanning confocal microscope (Leica TCS SP2) with a ×40/1.25 NA oil immersion objective lens. The plane of focus was made to bisect the centre of individual cells.
For visualisation of F-actin structure in rounded cells, the following procedure was performed. Transfected cells with LifeAct-GFP or actin-GFP were detached using trypsin, suspended in imaging media and fixed in 4% PFA for 10 min, followed by permeabilisation in 0.5% Triton X-100/PBS (Sigma-Aldrich) for 5 min prior to staining with Alexa Fluor 555-phalloidin (1:40; Invitrogen) in PBS+0.1% BSA for 20 min. Cells were then washed in PBS and suspended in distilled water. A drop of stained cells in suspension was placed on a coverslip and allowed to dry. Coverslips with cells were mounted using ProLong Gold and imaged using a laser scanning confocal microscope (Leica TCS SP2) with a ×40/1.25 NA oil immersion objective lens. The plane of focus was made to bisect the centre of individual cells.
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!