The largest database of trusted experimental protocols

5 protocols using prolong gold containing 4 6 diamidino 2 phenylindole dapi

1

Immunocytochemistry of BDNF-treated Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cultured cells were treated with BDNF for the indicated times, washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were permeabilized with PBS/0.5%Triton X-100 and then blocked for 1 hr with PBS/1% BSA/5% normal goat serum and exposed to primary antibodies overnight at 4°C. Cells were washed 3X with PBS, and exposed to secondary antibodies coupled to different fluorophores for 1 hr at room temp. Cells were washed three times in PBS and then mounted using Prolong Gold containing 4′,6′-diamidino-2-phenylindole (DAPI) (Life Technologies P36934). Cells were analyzed by epifluorescence (Nikon Eclipse TE200), confocal (Zeiss 510 Meta), or enhanced resolution (Leica SP8, 63X, 1.4 NA, Lightning Mode) microscopy. No immunostaining was seen in controls with omission of the primary antibodies.
+ Open protocol
+ Expand
2

Assessing Apoptosis in Osteogenic Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
MPC2 cells and hMSCs were plated on sterile glass chamber slides (Fisher Scientific) at 1.5 × 105 cells/mL. Once the cells reached 70% confluency the medium was changed to the appropriate corresponding osteogenic media and increasing concentrations of DFO. At the indicated time points, the media was removed from the cells and the wells were washed with PBS. The slides were fixed in 10% buffered formalin and underwent fluorescent terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL; Sigma) according to the manufacturer's instructions. DNaseI (Thomas Scientific) treatment was used as a positive control for staining as suggested by the manufacturer (Supplementary information Fig. S1). Coverslips were mounted with ProLong Gold containing 4,6‐diamidino‐2‐phenylindole (DAPI; Life Technologies) and four images per chamber were captured using a Leica fluorescent microscope. ImageJ software (NIH; https://imagej.nih.gov/ij/) was used to quantify DAPI‐positive and TUNEL‐positive cells. Each condition was conducted in two different chambers and the experiment was performed at least three times.
+ Open protocol
+ Expand
3

Indirect Immunofluorescence Microscopy Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Indirect immunofluorescence was conducted as described previously [9 (link)]. Briefly, seeded cells were fixed, permeabilized, blocked, then incubated overnight at 4 °C with primary mouse antibodies: HA (H9658, Sigma-Aldrich, Oakville, ON, Canada, 1:1000) or γ-H2A.X (ab26350, abcam, UK 1:1000). The next day, cells were washed and then incubated for 1 h in the dark with secondary antibody mouse Alexa 647 (Invitrogen, Life Technologies, Burlington, ON, Canada, 1:1000). Coverslips were mounted with ProLong Gold containing 4′,6′-diamidino-2-phenylindole (DAPI) (Invitrogen, Life Technologies). Images were taken at 40× magnification with an Olympus BX51 microscope using Image-Pro Plus (v5.0) software (Media Cybernetics, Inc., Bethesda, MD, USA). All indirect immunofluorescence figures were generated using ImageJ (v2.1.0) and zoomed-in; single-cell panels were generated using the QuickFigures plug-in [29 (link)].
+ Open protocol
+ Expand
4

Immunofluorescence Microscopy of GFP-Expressing Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Briefly, after permeabilization in a solution containing PBS, 0.1% Triton X-100 and 1% BSA (Sigma-Aldrich), cells were incubated with a primary antibody anti-GFP raised in goat (1:500, Rockland, Houston, TX, USA), followed by an incubation with a secondary antibody conjugated to AlexaFluor® 555 (1:1000, Invitrogen, Carlsbad, CA, USA). Slides were mounted with Prolong Gold containing 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen). The number of GFP-positive colonies was counted by epifluorescence microscopy and expressed as transduction units (TU) per mL. Knee joint sections with 10 μm thickness were obtained in a cryostat and mounted onto Superfrost slides (Thermo Fisher Scientific). After permeabilization, the sections were incubated with a primary antibody anti-GFP raised in rabbit (1:1000; Invitrogen) overnight at 4 °C, and then processed as described above. Images were acquired using a Zeiss LSM-710 confocal microscope (Carl Zeiss MicroImaging, GmbH, Jena, Germany).
+ Open protocol
+ Expand
5

Immunofluorescence Imaging of HA-tagged Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were seeded onto coverslips, fixed using 4% paraformaldehyde (PFA), then permeabilized with 0.5% Triton X-100 for 15 min at room temperature (RT). Cells were incubated overnight at 4 °C with primary mouse antibody against HA (H9658, Sigma-Aldrich, Oakville, ON, Canada, 1:1000) and then with secondary Alexa 488 antibody against mouse (Invitrogen, Life Technologies, Burlington, ON, Canada, 1:1000). The coverslips were mounted using ProLong Gold containing 4′,6′-diamidino-2-phenylindole (DAPI) (Invitrogen). Cell images were taken with an Olympus BX51 microscope at 40× magnification and Image-Pro Plus (v5.0) software (Media Cybernetics, Inc., Bethesda, MD, USA).
Mean nuclear intensities for immunofluorescence images of each BioID2 construct were determined using ImageJ (v2.1.0). For Regions of Interest (ROI), area and mean pixel intensities (scored between 0 and 255) were acquired by overlaying the binary DAPI image outline mask onto the HA fluorescence image and then averaging the mean ROI intensities and subtracting the background signal acquired from the HEK293 negative control cell line.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!