Prolong diamond antifade with dapi

Manufactured by Thermo Fisher Scientific

Sourced in United States

ProLong Diamond Antifade with DAPI is a mounting medium designed for fluorescence microscopy. It contains an antifade reagent to reduce photobleaching and DAPI for nuclear counterstaining.

Automatically generated - may contain errors

Lab products found in correlation

61 protocols using prolong diamond antifade with dapi

BrdU-based Proliferation Assay in Bone and Intestinal Tissues

Check if the same lab product or an alternative is used in the 5 most similar protocols

To assess cell proliferation, frozen bone sections were rehydrated in PBS, and paraffin ileum sections were deparaffinized and rehydrated in graded alcohol. A 1% trypsin in PBS solution was placed on the tissue for 10 min at 37°C. Slides were rinsed in PBS followed by 4 N HCl for 30 min at room temperature. Slides were rinsed in PBS then blocked in mouse‐on‐mouse blocking reagent (Vector Labs, Burlingame, CA, USA) for 10 min. A biotinylated monoclonal BrdU antibody (B35138, diluted 1:50 in PBS; Thermo Fisher Scientific, Waltham, MA, USA) was placed on the tissue and incubated overnight at 4°C. Frozen sections were brought to room temperature, rinsed in 1X PBS, and incubated with a Alexa Fluor 488 Streptavidin conjugate (S11223, diluted 1:100 in PBS; Thermo Fisher Scientific) for 1.5 hours. Sections were then rinsed and cover‐slipped with Prolong Diamond Antifade with DAPI (P36966; Thermo Fisher Scientific). Following antibody treatment, paraffin ileum sections proceeded with a HRP‐streptavidin and DAB substrate reaction and were counterstained with hematoxylin. For paraffin histology, bright‐field BrdU images were obtained using the Zeiss Axio Imager Z2 microscope and a 20× objective. For frozen histology, fluorescent images were obtained along the periosteal compressive surface on the Zeiss Axio Imager Z2 with the GFP filter used to detect BrdU Alexa 488.

Zannit H.M, & Silva M.J. (2019). Proliferation and Activation of Osterix‐Lineage Cells Contribute to Loading‐Induced Periosteal Bone Formation in Mice. JBMR Plus, 3(11), e10227.

+ Open protocol

+ Expand

Antibodies and Reagents for Mitochondrial Studies

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following antibodies were used in this study: rabbit anti-Myo19 (HPA059715, 1:1000 for western blotting) from Sigma-Aldrich; rabbit anti-Mic60 (A2751, 1:3000 for western blotting), anti-Metaxin2 (A7958, 1:1000 for western blotting) and rabbit anti-Sam50 (A3401, 1:3000 for western blotting, 1:400 for PLA assay) from ABclonal; mouse anti-Mic60 (sc-390707, 1:200 for immunofluorescence staining) from Santa Cruz Biotechnology; rabbit anti-GFP (598, 1:1000 for western blotting, 1:200 for immunofluorescence staining) and mouse anti-GFP (MO48-3, 1:200 for immunofluorescence staining) from MBL; anti-mouse (sc-516102, 1:4000) and anti-rabbit (sc-2004, 1:4000) horseradish peroxidase (HRP)-conjugated secondary antibodies from Santa Cruz Biotechnology; Alexa Fluor488- or Alexa Fluor 555-conjugated secondary antibody from ThermoFisher Scientific. For reagents, MitoTracker^TM Red CMXRos (M7512), MitoTracker^TM Green FM (M7514), TMRE (T669), TMRM (T668) and Prolong Diamond Antifade with DAPI (P36962) were purchased from Thermo Fisher Scientific. Rotenone (HY-B1756), FCCP (HY-100410) and 2-Deoxy-D-glucose (HY-13966) were purchased from MedChemExpress.

Shi P., Ren X., Meng J., Kang C., Wu Y., Rong Y., Zhao S., Jiang Z., Liang L., He W., Yin Y., Li X., Liu Y., Huang X., Sun Y., Li B, & Wu C. (2022). Mechanical instability generated by Myosin 19 contributes to mitochondria cristae architecture and OXPHOS. Nature Communications, 13, 2673.

+ Open protocol

+ Expand

Formaldehyde-Fixed Cell Immunofluorescence

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were fixed for 15–20 min in methanol-free formaldehyde (Pierce), which was freshly diluted to 2% final concentration with PBS, pH 7.4. Fixed cells were washed three times with PBS, permeabilized and blocked with 0.3% Triton-X and 3% BSA in PBS for 1 hour at room temperature, incubated overnight at 4 °C with primary antibodies diluted in the same buffer, washed twice with PBS, incubated with secondary antibodies in 3% BSA in PBS for 30–60 minutes at room temperature, and mounted with Prolong Diamond Antifade with DAPI (ThermoFisher). We used the following antibodies: Rabbit anti-Col1 (AB765P, 1:400; Millipore), Mouse anti-GFP (GTX628528, 1:400; GeneTex), Goat anti-Rabbit IgG (DyLight 550, SA5-10033, 1:10,000; ThermoFisher), and Goat anti-Mouse IgG (AlexaFluor 647, A-21236, 1:10,000; ThermoFisher).

Gorrell L., Omari S., Makareeva E, & Leikin S. (2021). Noncanonical ER–Golgi trafficking and autophagy of endogenous procollagen in osteoblasts. Cellular and molecular life sciences : CMLS, 78(24), 8283-8300.

+ Open protocol

+ Expand

GLP-1R-Expressing Islet Cells Visualization

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Pancreatic islets were isolated from GLP-1R-iCre-GCaMP3 mice (9–13 weeks old), a mouse strain in which GCaMP3 is restricted to cells with GLP-1R promoter activity [22 (link)]. Pancreata were inflated with RPMI-1640 medium containing 1 mg/mL collagenase from Clostridium histolyticum (S1745602, Nordmark Biochemicals, Germany), dissected and incubated in a water-bath at 37 °C for 12 min. Islets were subsequently washed and purified using a Histopaque gradient (Histopaque-1119 and -1083). Isolated islets were allowed to recover overnight in RPMI-1640 supplemented with 10% and 1% penicillin/streptomycin. Islet dispersal was achieved by trituration in 0.05% trypsin-EDTA at 37 °C for 3 min, followed by trypsin inactivation with complete medium. Dispersed islet cells were then seeded onto poly-D-lysine-coated coverslips and allowed to adhere overnight in complete RPMI-1640. The following day, 100 nM GLP-1R-TMR, exendin-4-TMR, or vehicle were added to the well, and incubated for 30 min at 37 °C. Medium was then removed, and cells were washed once with HBSS and fixed with 4% PFA for 15 min. Washed coverslips were subsequently mounted onto glass slides in Prolong Diamond anti-fade with DAPI (Thermo Fisher).

Fang Z., Chen S., Manchanda Y., Bitsi S., Pickford P., David A., Shchepinova M.M., Corrêa Jr I.R., Hodson D.J., Broichhagen J., Tate E.W., Reimann F., Salem V., Rutter G.A., Tan T., Bloom S.R., Tomas A, & Jones B. (2020). Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells. International Journal of Molecular Sciences, 21(21), 8404.

+ Open protocol

+ Expand

Evaluating Bronchial and Liver Organoids

Check if the same lab product or an alternative is used in the 5 most similar protocols

Example 3

Determining Morphology of Bronchial Organotypic Cultures and Liver Spheroids

Morphology of bronchial organotypic cultures is evaluated following fixation and paraffin embedding, sectioning and staining with hematoxylin and eosin (H&E) and Alcian blue as previously described in Toxicol Sci. 2015 September; 147(1):207-21.

Liver spheroid morphology is assessed following immunostaining. In brief, liver spheroids are fixed in 4% fresh paraformaldehyde overnight. Following blocking in 1% Triton X-100/0.2% fish skin gelatin (FSG), spheroids are stained with mouse anti-cytokeratin 19 (1/500, Abcam, Cambridge, UK) diluted in PBS with 0.1% FSG for 24 hours. The primary antibody is visualized using a FITC-labeled goat anti-mouse antibody (1/500, Abcam). Spheroids are then mounted using ProLong Diamond antifade with DAPI (Thermo Fisher) and evaluated by high-content imaging on the Cellinsight™ CX7 platform (Thermo Fisher).

US11041145B2. Cell culture (2021-06-22). PHILIP MORRIS PRODUCTS S.A. [CH]. Inventors: David Bovard [CH], Karsta Luettich [CH].

+ Open protocol

+ Expand

Immunofluorescent Labeling of Embryonic Tissues

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells and tissue sections were fixed in 4% PFA (158127 Merck). Embryos were cryosectioned at 12 µm. Cryosections and fixed cells were blocked in 10% DAKO serum-free blocking solution (X0909 DAKO) in PBST. The following primary antibodies and stains were used: anti-SOX10 (AF2864 R&D, 1:200), anti-RUNX2 (12556 Cell Signalling, 1:250), Alexa-fluor 594 phalloidin (A12381 Life technologies, 1:1000). The following secondary antibodies were used at 1:200 dilution: anti-rabbit AlexaFluor 488 (A21206 Thermo Fisher); anti-rabbit AlexaFluor 555 (A31572 Thermo Fisher); anti-mouse AlexaFluor 647 (A31571 Thermo Fisher); anti-mouse AlexaFluor 488 (A21202 Thermo Fisher); anti-rat AlexaFluor 488 (A21208 Thermo Fisher). Tissue sections and cells were mounted in Prolong Diamond Anti-fade with DAPI (P36962 Thermo Fisher). Images were acquired using a Zeiss LSM 800 confocal microscope and ZEN (blue) software.

Lohraseb I., McCarthy P., Secker G., Marchant C., Wu J., Ali N., Kumar S., Daly R.J., Harvey N.L., Kawabe H., Kleifeld O., Wiszniak S, & Schwarz Q. (2022). Global ubiquitinome profiling identifies NEDD4 as a regulator of Profilin 1 and actin remodelling in neural crest cells. Nature Communications, 13, 2018.

+ Open protocol

+ Expand

Immunofluorescent Localization of Podocin Variants

Check if the same lab product or an alternative is used in the 5 most similar protocols

HeLa cells transiently expressing FLAG-tagged podocin^wildtype and podocin^Δexon5 were seeded on coverylips at 37 °C overnight. On the following day, the cells were fixed with 4% paraformaldehyde (PFA) for 8 min at room temperature after washing with phosphate buffered saline (PBS). Cells were blocked with 5% normal donkey serum in PBST for 30 min at room temperature and incubated with a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich Cat# F3165, RRID:AB_259529, 1:1000) at 4 °C overnight and a donkey anti-mouse cy3 secondary antibody (Jackson ImmunoResearch Labs Cat# 715–165-150, RRID:AB_2340813, 1:500) for 1 h at room temperature. Samples were mounted in ProLong Diamond antifade with DAPI (Thermo Fisher Scientific). An Axio Observer microscope with ZEN software version 2.6 (both Carl Zeiss, Germany) was used to acquire images that were further processed with ImageJ/Fiji software version 1.52i (NIH, Bethesda, MD, USA) [28 (link)].

Butt L., Unnersjö-Jess D., Reilly D., Hahnfeldt R., Rinschen M.M., Bozek K., Schermer B., Benzing T, & Höhne M. (2023). In vivo characterization of a podocyte-expressed short podocin isoform. BMC Nephrology, 24, 378.

+ Open protocol

+ Expand

Quantifying NETosis by Confocal Microscopy

Check if the same lab product or an alternative is used in the 5 most similar protocols

To confirm in vitro NETosis in the bacteria experiments, we added an immunofluorescent staining with a MPO-Dylight488 complex (1:250) to the neutrophils immediately before induction. Then, we quantified the positive NETs by using confocal microscopy (Leica SP5 AOBS).
As another measurement for NETosis, cells were stimulated by the described inducers for 3 hours, fixed and stained for myeloperoxidase (MPO, Dako). Briefly, after antigen retrieval with Proteinase K, the slides were blocked with skim milk powder (5%) in PBS Tween 0.1% pH7.4 and incubated overnight with polyclonal rabbit anti-human MPO (1:300) at 4°C. After washing with PBS Tween 0.1% pH7.4 slides were incubated with secondary antibody Dylight goat anti rabbit 488 (1:200) for 30 minutes. Slides were mounted with Prolong Diamond antifade with DAPI (Thermofischer). Images were made by using confocal microscopy (Leica SP5 AOBS) and Structured Illumination Microscopy (Zeiss Elyra PS1 LSM 780 structured illumination microscope, Carl Zeiss, Jena, Germany).

Hoppenbrouwers T., Autar A.S., Sultan A.R., Abraham T.E., van Cappellen W.A., Houtsmuller A.B., van Wamel W.J., van Beusekom H.M., van Neck J.W, & de Maat M.P. (2017). In vitro induction of NETosis: Comprehensive live imaging comparison and systematic review. PLoS ONE, 12(5), e0176472.

+ Open protocol

+ Expand

Immunofluorescent Staining of p-MLKL in Lm-infected or TNF/CHX-stimulated HepG2 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

HepG2 cells plated on a coverslip were infected with Lm or stimulated with TNF/CHX as described before. Following stimulation, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and thereafter permeabilized with 0.1% Triton-X-100 for 7 min. Nonspecific bindings were blocked by incubating the cells for 45 min in 0.5% BSA, 0.3% Triton-X-100, 5% sucrose, 10% fetal calf serum) before staining the cells with anti-p-MLKL (Cat# ab187091, Abcam, Cambridge, UK) overnight on a rocking platform. The slides were washed three times with PBS and then incubated with the fluorochrome-conjugated secondary antibody (Goat Anti-Rabbit IgG, Alexa Fluor Plus 594, Thermo Fisher Scientific, Waltham, USA) for 2 h protected from light. After washing with PBS, the slides were mounted on a glass slide using ProLong Diamond Antifade with DAPI (Cat# P36962, Thermo Fisher Scientific, Waltham, USA). Images were captured on Microscope Axio Imager Z1 (Carl Zeiss AG, Oberkochen, Germany).

Koschel J., Nishanth G., Just S., Harit K., Kröger A., Deckert M., Naumann M, & Schlüter D. (2021). OTUB1 prevents lethal hepatocyte necroptosis through stabilization of c-IAP1 during murine liver inflammation. Cell Death and Differentiation, 28(7), 2257-2275.

+ Open protocol

+ Expand

Reagents for Cell Imaging Assays

Check if the same lab product or an alternative is used in the 5 most similar protocols

Drugs and dyes used in this
study were obtained from commercial vendors. CAS numbers are as following:
Sorafenib (284461-73-0) from Synchem, Inc.; Fulvestrant (129453-61-8)
from Sigma; Lapatinib (388082-78-8) from Larid Road; Clofazimine (2030-63-9)
from Sigma; Tartrazine (1934-21-0), and Light green SF yellowish (5141-20-8)
from Alfa Aesar. Alexa fluor WGA555 and Prolong Diamond Anti-fade
with DAPI was purchased from Thermo Fisher Scientific.

Dlim M.M., Shahout F.S., Khabir M.K., Labonté P.P, & LaPlante S.R. (2019). Revealing Drug Self-Associations into Nano-Entities. ACS Omega, 4(5), 8919-8925.

+ 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?

Revolutionizing how scientists
search and build protocols!