For confocal microscopy imaging, transfected cells and lupus patient peripheral blood mononuclear cells were stained with the indicated antibodies, including LC3B (Invitrogen, PA5-30598), TOMM20 (EPR15581-39, Abcam, ab205487), and Parkin (clone PRK8, Santa Cruz Biotechnology, sc-32282), and sorted by BD FACSAria. Cells were resuspended in PBS and allowed to be adhered to cover slide coated with Corning Cell-Tak Cell and Tissue Adhesive (Fisher Scientific, CB-40240). Cover slides were then mounted with ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, P36931). Confocal microscopy was done with a Zeiss LSM 880 upright confocal microscope at Confocal Imaging and Immunohistochemistry Core Facility, Beth Israel Deaconess Medical Center.
Prolong gold antifade mountant with 4 6 diamidino 2 phenylindole dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States
ProLong Gold Antifade Mountant with 4',6-diamidino-2-phenylindole (DAPI) is a fluorescent mounting medium designed for preserving and protecting fluorescent signals in microscopy samples. The product contains DAPI, a nuclear counterstain that binds to DNA, allowing visualization of cell nuclei. The antifade properties of the mountant help maintain the fluorescent signals over time.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Hrec ade180sur 03, by Shanghai Outdo Biotech Co. (2 mentions)
Alexa fluor 549 mouse anti cd3, by BioLegend (2 mentions)
Dylight 488rabbit anti tigit, by BioLegend (2 mentions)
Leica aperio system, by Leica camera (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Uplansapo 60x objective, by Olympus (2 mentions)
8 well culture chamber slides, by Thermo Fisher Scientific (2 mentions)
Lyso dye, by Thermo Fisher Scientific (2 mentions)
Fluoview clsm, by Olympus (2 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
Tomm20, by Abcam (1 mentions)
Lsm 880 upright confocal microscope, by Zeiss (1 mentions)
Facsaria, by BD (1 mentions)
Confocal laser scanning microscope, by Leica camera (1 mentions)
Lab tek 2 chamber slide system, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
51 protocols using prolong gold antifade mountant with 4 6 diamidino 2 phenylindole dapi
1
Confocal Imaging of Autophagy Proteins
2
MCMV-M45mutRHIM Infection Assay with ThT Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary tail fibroblasts were seeded onto an 8-well chamber slide (Nunc Lab-Tek II Chamber Slide system) for overnight culture at 37 °C and infected with a m.o.i. = 5 of MCMV-M45mutRHIM mutant virus for the indicated times. ThT (25 μM) was added to cells 1 h before fixation18 (link). Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized in 0.1% Triton X-100 in PBS for 10 min and blocked with 0.5% BSA in PBS containing 0.1% saponin at room temperature. Cells were then incubated with the indicated antibodies overnight at 4 °C, followed by washing with PBS-T three times at room temperature and incubation with anti-mouse IgG Cascade Blue, anti-mouse IgG Alexa Fluor 568, anti-rabbit IgG Alexa Fluor 488 or anti-rabbit IgG Alexa Fluor 568 (Thermo Fisher) at 1:1,000 dilution. The coverslips were mounted and counterstained using ProLong Gold Antifade mountant with 4,6-diamidino-2-phenylindole (DAPI, Thermo Fisher). All images were captured using identical settings on a Nikon laser scanning confocal microscope or a Leica SP8 confocal microscope with a ×60 oil-objective, and processed using Nikon’s NIS elements, Leica’s LAS X and ImageJ software.
3
Immunofluorescence Localization of MT1 and MT2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NCI-H1703, A549 and IMR-90 cell lines were set up 2 × 104 cells per well Millicell EZ 8-wells glass slide (Merck, Darmstadt, Germany, Cat# PEZGS0816) in appropriate medium overnight. The cells were fixed by 12 min incubation in 4% paraformaldehyde at room temperature (RT). The membranes were permeabilized using 0.2% Triton (10 min/RT). Sites of non-specific binding were blocked using 3% BSA in 0.1% TBST buffer (1 h/RT). The cells were incubated for 1h at RT with primary MT1-specific antibodies, diluted 1:3200 (Invitrogen, Carlsbad, CA, USA) in 1% BSA in PBS and MT2-specific antibodies diluted 1:100 in 1% BSA in PBS (Abcam, Cambridge, UK, Cat# ab203346, RRID:AB_2783824). Subsequently, secondary anti-rabbit antibodies Alexa 568 were applied (1:2000, 1h/RT; Abcam, Cambridge, UK, Cat# ab175470, RRID:AB_2783823). Negative controls were performed with 1% BSA in PBS instead of the specific antibody. The preparations were mounted in a ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (ThermoFisher, Waltham, Massachusetts, USA). A Confocal Laser Scanning Microscope Fluoview FV3000 (Olympus, Hamburg, Germany, RRID:SCR_017015) coupled with CellSense software (Olympus, Hamburg, Germany, RRID:SCR_016238) was used for fluorescence.
4
Visualizing Neutrophil Extracellular Traps
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutrophils (5×105) were seeded into IBIDI™ µ-slide 8-well chamber slides, pre-incubated for 1 h with ruboxistaurin (200 nM), and NETosis was induced by LPS and PMA as described above. Cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. Wells were blocked and permeabilised with buffer containing 5% bovine serum albumin (BSA), 0.1% saponin and 5% normal goat serum, and then stained with a rabbit anti-MPO (A0398) primary antibody for 90 min at 37°C. A secondary goat anti-rabbit Alexa Fluor® 594 antibody (ab150088) was added for 45 min at 37°C. ProLong™ Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher) was used to stain DNA. Samples were imaged using a NIKON Widefield fluorescence microscope, using the 40× oil immersion objective lens. The DAPI (excitation/emission 395/455 nM) and Texas Red (excitation/emission 555/605 nM) filter sets were used for fluorescent imaging. Images were constructed using FIJI image analysis software, and the background was subtracted for the DAPI channel using FiJI.
5
Immunofluorescent Localization of Cardiac Progenitor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human myocardial samples were either fixed in formalin and paraffin-embedded, or frozen using OCT compound. Five-micrometre-thick sections were cut for identification of cardiac PCs in situ. Paraffin sections required heat-induced antigen retrieval, performed using citrate buffer 0.01 M pH = 6, for 40 min at 98°C. Tissue sections were blocked with 10% v/v normal donkey serum and incubated with primary antibodies for 16 h at 4°C. Antibodies were: anti-CD34 (Dako M7165, 1:100), anti-CD31 (Abcam ab28364, 1:50), anti-platelet derived growth factor receptor β (PDGFRβ – R&D AF385, 1:50), anti-smooth muscle α-actin (α-SMA – Dako GA611, 1:100), anti-ACE2 (Merck SAB3500346, 1:40), anti-CD147 (BioLegend 306221, 1:100), anti-von Willebrand Factor (vWF – Merck F3520, 1:200). Donkey secondary antibodies (Alexa 488-, Alexa 568-, Alexa 647-conjugated) were all purchased from Thermo Fisher Scientific and used at a dilution of 1:200, for 1 h at 20°C in the dark. Slides were mounted using ProLong™ Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific). Imaging was performed using a Leica TCS SP8 confocal microscope and a Leica SP5-II AOBS multi-laser confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope (Leica Microsystems).
6
Colorectal Cancer Tissue Microarray Immunofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue microarrays (HRec-Ade180Sur-03, Shanghai Outdo Biotech Co.) were constructed using formalin-fixed, paraffin-embedded carcinoma tissues matched to adjacent normal tissues collected from 90 colorectal cancer cases (cohort 2). Immunofluorescence was performed following an established protocol. Briefly, tissue microarrays were dewaxed, rehydrated through graded ethanol, and incubated with 3% hydrogen peroxide for 30 mins. Antigen retrieval was performed by heating the sections to 95 °C in 0.01 M citrate buffer (pH6.0) for 15 mins. Slides were then washed in PBS for 15 mins, treated with 10% normal horse serum for 30 mins and incubated with the primary antibodies, including Alexa Fluor 549 mouse anti-CD3 (BioLegend, San Diego, USA) and DyLight 488rabbit anti-TIGIT (BioLegend, San Diego, USA). After staining, the slides were mounted with ProLong Gold Antifade Mountant with 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher, USA). The images were taken using the Leica Aperio System (Leica, USA).
7
Tracking EV Uptake in Aged MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MSCs (2.5 × 105) from old and young individuals were cultured on slides (Sigma-Aldrich, St. Louis, MO, USA) pre-treated with poly-d -lysine (Sigma-Aldrich, St. Louis, MO, USA) in 6-well plates (Corning Inc., New York, NY, USA) for 8 h (day 1). MSC-derived EVs (2 × 107) from young and old individuals were stained with 10 μM 3-3′-diethylthiacarbocyanineiodide (DiI) and added to each culture of MSCs; PBS (MP Biomedicals, Illkrich-Graffenstaden, France) was added as a control. At 2, 3 and 6 days, the cells were washed three times with PBS (MP Biomedicals, Illkrich-Graffenstaden, France) and fixed with 4% (w/v) paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 10 min, and then the slides were mounted using ProLong® Gold antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI; (Thermo Fisher Scientific, Carlsbad, CA, USA). The cells were examined with an Olympus BX61 microscope using a DP71 digital chamber (Olympus, Tokyo, Japan) with software DP-Controller and DP-Manager software. A NeoScope JCM-6000 Plus scanning electron microscope (Nikon-Izasa, Barcelona, Spain) was used to take supplementary images.
8
Cytoskeletal Analysis of Colon Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fifty thousand HT-29, DLD-1, or SW-480 cells were seeded in 24-well plates with coverslips inserted. The serum starvation and incubation under different oxygen levels with or without JNK inhibitor SP600125 was the same as that of the cell morphological analysis. After incubation, cells were first gently washed by phosphate-buffered saline (PBS) for three times, then fixed with 4% paraformaldehyde (PFA) in PBS for 30 min. After washing by PBS for another three times, the cells were blocked with 2% bovine serum albumin (BSA) in PBS for 30 min. Alexa Fluor 594 Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) was added to the cells for staining F-actin in 1 h. Cells were mounted with ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) after PBS washing. Cells and their cytoskeletons were visualized by confocal microscopy (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany). The effect of oxygen level on cytoskeleton was quantified in terms of lamellipodia, filopodia, and ventral stress fibers. At least 200 cells were scored in each of the three independent experiments, and the percentages of cells having the three cytoskeleton structures of different conditions were plotted and compared.
9
Immunofluorescence Staining of PD-L1 and NK Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PDL1 t-haNK, MDA-MB-231 and PD-L1 null MDA-MB-231 cells were stained as described.9 (link) The following were used as primary antibodies: NKG2d (Abcam) and perforin (Abcam), PD-L1 (Abcam). The following were used as secondary antibodies: goat anti-rabbit-AlexFLuor647 (Thermo Fisher), goat anti-mouse-AlexFLuor488 (Thermo Fisher), and goat-anti-rabbit AlexaFluor488 (Thermo Fisher). The samples were mounted using ProLong gold antifade mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher). The samples were imaged on a Leica DMI4000B microscope (Leica Microsystems).
10
Imaging of Transfected C2C12 and Fibroblast Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C2C12 cells were transfected with either eGFP-LEMD2 or mCherry-Lamin A/C or co-transfected with both by using Lipofectamine 2000 (Thermo Fisher Scientific). Forty-eight hours after transfection, cells were fixed for 20 min in 4% paraformaldehyde at 4°C, washed 3 times with phosphate-buffered saline (PBS) for 10 min each) and blocked for 1 h with goat serum. Afterwards, the slides were washed, and Prolong Gold Antifade Mountant with 4′,6′-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) was added. Fibroblast cells were spread on 0.01% gelatin-coated cover slides and incubated at 37°C for 48 h and fixed with 4% paraformaldehyde for 20 min at 4°C. Paraffin-embedded tissue slides (5 μm) were deparaffinized by using xylene and ethanol. Tissue and fixed fibroblast cells were washed with PBS, blocked with goat serum, and stained overnight with primary antibodies followed by secondary antibodies conjugated with Alexa-488 and Alexa-555 dyes for 2 h at room temperature (Supplemental Table 1 and Supplemental Figure 1 for isotype control staining) and embedded in ProLong Gold Antifade Mountant with DAPI. The LSM5 Exciter (Carl Zeiss AG, Oberkochen, Germany) was used for confocal imaging. All images were processed with Zen software v6,0,0,303 (MDaemon Technologies, Ltd., Grapevine, Texas).
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!