Reagents and the detailed protocol for smFISH and IF were described in Lyubimova et al. (2013) (link). Briefly, cells were fixed with 4% PFA (diluted in 1× PBS), permeabilized with permeabilization buffer (1× PBS, 5 mM MgCl2, 0.5% Triton), and incubated with prehybridization buffer (2× SSC, 15% formamide). Cells were incubated with fluorescently labeled PLK4-Cy3 (80 nM) smFISH probes and rabbit DDX4 (1:250; Abcam ab13840) antibody diluted in hybridization buffer for 3 h at 37°C. Oligonucleotide probe sequences are listed in Supplemental Table S2 . Following washes, cells were incubated with Alexa fluor 488 secondary antibody (Life Technologies) diluted in hybridization buffer for 1 h at room temperature. Cells were mounted using ProLong Diamond antifade reagent with DAPI (Life Technologies). For single-molecule mRNA visualization, images were collected using a Leica 63×, 1.40 NA oil with 0.2-μm z-sections.
Prolong diamond anti fade reagent with dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States
ProLong Diamond Anti-fade reagent with DAPI is a mounting medium designed for fluorescence microscopy. It contains a fluorescent dye, DAPI, which binds to DNA and emits blue fluorescence. The reagent also includes an anti-fade compound to reduce photobleaching of fluorescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Microm hm550 cryostat, by Thermo Fisher Scientific (6 mentions)
Coverslip, by Thermo Fisher Scientific (3 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Anti mouse af647, by Thermo Fisher Scientific (2 mentions)
Anti rabbit af647, by Thermo Fisher Scientific (2 mentions)
Lsm880 fast airyscan confocal microscope, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Tritc conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Ab13840, by Abcam (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Axioskop 2 microscope, by Zeiss (1 mentions)
Anti brdu antibody, by Merck Group (1 mentions)
Formaldehyde, by Thermo Fisher Scientific (1 mentions)
5 fluorouridine, by Merck Group (1 mentions)
Sola light engine, by Hamamatsu Photonics (1 mentions)
Orca r2 digital ccd camera, by Hamamatsu Photonics (1 mentions)
Cy3 4040c zero, by IDEX Corporation (1 mentions)
Dapi 5060c zero, by IDEX Corporation (1 mentions)
23 protocols using prolong diamond anti fade reagent with dapi
1
Visualization of PLK4 and DDX4 in Cells
2
Immunofluorescent Imaging of Plasmodium Infection
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An. stephensi midguts were dissected on day 12 post-infection and fixed in 4% paraformaldehyde for 1 h on ice or overnight at 4°C. Midguts were incubated with 3% bovine serum albumin (BSA) and 0.1% Triton-X-100 to block and permeabilize for 1 h at room temperature or overnight at 4°C. Samples were washed in PBS and incubated overnight at 4°C in a 1:1,000 dilution of 0.5 mg/ml mouse anti-P. falciparum circumsporozoite protein [PfCSP; 2A10; MRA183A, Malaria Research and Reference Reagent Resource Center (MR4), Bei Resources], in 3% BSA. Following incubation with the primary antibody, midguts were washed in PBS and incubated for 1 h in a 1:1,000 dilution of 2 mg/ml Alexa Fluor goat anti-mouse 594 antibody (Life Technologies, Frederick, MD) in 3% BSA while being protected from light. Following incubation with the secondary antibody, midguts were washed in PBS, deposited on a slide in a drop of ProLong Diamond Antifade Reagent with DAPI (Life Technologies, Frederick, MD), and covered with a 22 × 22 mm coverslip. Slides were viewed and imaged using a Nikon Upright E800, Nikon 90i, or Zeiss Axioskop2 microscope and the GNU Image Manipulation Program (GIMP).
3
AICAR-Induced 5-Fluorouridine Labeling
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10,000 cells were plated onto a 12 mm glass cover slip in a 24-well plate and incubated for 2 nights. The cells were treated with 1 mM AICAR for 12 hrs and then labeled with 2 mM 5-fluorouridine (Sigma) for 15 mins in the incubator. The coverslips were washed with PBS and fixed in 2% formaldehyde (Thermo Fisher Scientific Inc.) for 20 mins in the dark at room temperature. The fixed cells were permeabilized with methanol (Thermo Fisher Scientific Inc.) at −20°C for 10 mins. Coverslips were then blocked with 2% goat serum (Sigma) for 30 mins and incubated with the anti-BrdU antibody (Sigma) at 4°C overnight. The coverslips were washed with PBS and incubated with a secondary goat anti-mouse IgG/IgM (H+L) AlexaFluor® 488 conjugated antibody (Life Technologies) for 1 hr at room temperature. After washing with PBS 3 times, cells were mounted using the Prolong Diamond anti-fade reagent with DAPI (Life Technologies). Images were obtained as single optical slides using a LSM510-Meta confocal microscope (Zeiss, Jena, Germany) equipped with a 63×1.3 oil immersion objective and excitation wavelengths of 405 nm and 488 nm.
4
Quantifying Arc Expression in Hippocampal Neurons
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Hippocampal neurons plated at 60,000 cells/Mattek dish were transduced with Suntag-Arc translation reporter for 10 days were stimulated with TTX-washout paradigm at DIV 19 and fixed. The neurons were then permeabilized and smFISH-IF was performed according to the protocol described in (Eliscovich et al., 2017) . Briefly, 100nM probes against 24X Suntag sequence and primary antibodies against GCN4 epitopes (Clone C11L34, Ab00436-1.4, Absolute antibody, Wilton, UK) was used in hybridization buffer for 3 hours at 37ºC. After washes, the cells were incubated with Alexa Fluor 647 secondary antibody (Life Technologies) and mounted using ProLong diamond antifade reagent with DAPI (Life Technologies). Images were taken in a custom up-right widefield Olympus BX-63 microscope equipped with a Lumencor SOLA-Light engine, ORCA-R2 Digital CCD camera (Hamamatsu), SuperApochromatic 60x/1.35 NA Olympus objective (UPLSAPO60XO) and zero pixel shift filter sets: DAPI-5060C-Zero, Cy3-4040C-Zero and Cy5-4040C-Zero (Semrock). Image pixel size: XY, 107.5 nm; Z-steps, 200 nm. The sequences for FISH probes have been described in (Wu et al., 2016) .
5
Exosome Labeling and Uptake Visualization
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Twenty-four μg of exosomes were incubated with lipophilic tracer DiO solution (Thermo Fisher Scientific) for 20 min at 37 °C. Excessive DiO was removed with Exosome Spin Columns (MW 3000) (Thermo Fisher Scientific). Exosome labeling efficiency was analyzed with an Infinite® 200 PRO fluorometer (TECAN, Männedorf, CHE). The cells were seeded in an 8-well chamber slide (1 × 104 or 4 × 104 cells/well) and incubated for 24 h. DiO-labeled exosomes (8 μg) were added to the culture media of the recipient cells and incubated for 3 h at 37 °C. The recipient cells were fixed with 4% paraformaldehyde at room temperature for 10 min and permeabilized with 0.1% Triton X-100 at room temperature for 5 min. The cells were stained with Alexa Fluor 555 phalloidin (Thermo Fisher Scientific) at room temperature for 30 min and mounted in Prolong® Diamond Antifade Reagent with DAPI (Thermo Fisher Scientific), and the slide was covered with cover glass. The cells were visualized with an EVOS FL fluorescence microscope (Thermo Fisher Scientific).
6
Immunofluorescence Staining of Brain Sections
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Frozen mouse brain sections were blocked with 5% goat serum in PBS, and cells were then stained with the indicated primary antibodies for 1 h at room temperature. After washing, the cells were stained with secondary antibodies for 1 h at room temperature, then mounted in Prolong Diamond Antifade reagent with DAPI (Thermo Fisher Scientific), and observed using a laser‐scanning confocal microscope (LSM710, Zeiss). Paraffin sections of a patient's brain were deparaffinized in xylene, rehydrated through a graded series of ethanol, and then treated as described above for mouse brain sections.
7
Immunofluorescence Staining of Pancreatic Cancer Cells
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Cells of each cell line (ASPC-1, SUIT-2, and PANC-1) were plated on coverslips in 12-well plates and cultured for 24 hr. The cells were then washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 10 min, and washed three times in PBS. The cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min, and washed three times. A 15-min incubation with the reagent Blocking One-P (Nacalai Tesque) was followed by an overnight incubation using the primary antibody FAM115C (1:200, orb183515; Biorbyt, Cambridge, UK) diluted with 10% Blocking One-P in PBS at 4℃. The coverslips were then washed three times with PBS and incubated for 40 min at room temperature with Alexa 488-labeled donkey anti-mouse (1:1000; Invitrogen) diluted with 10% Blocking One-P in PBS. The wells were washed three times in PBS and then mounted on glass coverslips on a microscope slide with Prolong Diamond Antifade Reagent with DAPI (Thermofisher Scientific, Waltham, MA). Images were visualized using a confocal microscope (A-1; Nikon).
8
Immunofluorescence Staining of Cultured Cells
9
Immunostaining of Cryosectioned Livers
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Dissected livers were fixed in 4% PFA overnight at 4°C and washed with PBS/0.1% Tween 20 before incubation in 30% sucrose in PBS overnight at 4°C. Livers were aligned in a tissue mould, embedded in OCT and frozen on dry ice. The livers were sectioned at 10µm intervals using a Thermo Fisher Scientific Microm HM550 cryostat. Sections were washed with PBS before blocking with 10% FCS in PBS/0.3% Triton X-100. Incubation with primary antibodies was performed at 4°C overnight, while incubation with secondary antibodies was performed at room temperature for 1 hr. Antibodies used in this work were: 1:2000 a-Tubulin DM1A (CST, #3873), 1:1000 γ-H2AX (gift of James Amatruda), 1:250 cleaved caspase-3 (CST, #9664), 1:500 anti-rabbit AF647 (Thermo Fisher Scientific, #A31573) and 1:500 anti-mouse AF647 (Thermo Fisher, #A21235). Prolong Diamond Antifade reagent with DAPI (Thermo Fisher #P36962) was used for slide mounting. A Zeiss LSM880 Fast Airyscan Confocal microscope with a ×63 objective was used for image acquisition and ImageJ for image analysis.
10
Embryo Immunofluorescence in Drosophila
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We performed embryo immunofluorescence of fixed, staged embryos after aging embryos laid on grape juice plates to 2–4 hours, which we then dechorionated in 50% bleach and collected using a 40micron strainer. We immediately fixed embryos using 37% formaldehyde in heptane for 20 minutes. We then collected and washed embryos in methanol and stored in −20°C. We began immunostaining by rehydrating embryos using increasing concentrations of PB-Tween in Methanol. We then washed embryos and incubated with primary antibody in block (1% BSA in 1x PBS) overnight at 4°C on a rotator. The following day, we collected embryos and washed in block before incubating with secondary antibody for 2 hours at room temperature protected from light. We then washed and mounted embryos on slides using Prolong Diamond anti fade reagent with DAPI (ThermoFisher Scientific, P36961). We imaged embryos using a Keyence BZ-X810 Fluorescence microscope using a 20x objective. We conducted Image processing using ImageJ software (NIH).
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