A commercially available NEAT1 probe (Stellaris FISH probe for human NEAT1, 5′ segment; Biosearch Technologies) was used as per the manufacturer's protocol. Briefly, cells were fixed with 4% PFA for 15 min, followed by permeabilization with 0.5% Triton X‐100 for 5 min. Cells were incubated in 10% formamide/2× SSC for 10 min at RT followed by hybridization at 37°C for 16 h. Samples were mounted in ProLong Diamond antifade reagent (Thermo Fisher). Quantification was performed using ImageJ software (National Institutes of Health).
Prolong diamond antifade reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom
ProLong Diamond antifade reagent is a mounting medium designed to preserve fluorescent signals in microscopy samples. It helps to minimize photobleaching, allowing for longer observation and imaging of fluorescently labeled specimens.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (16 mentions)
Triton x 100, by Merck Group (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (8 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (7 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (6 mentions)
Paraformaldehyde (pfa), by Merck Group (6 mentions)
Lsm 780 confocal microscope, by Zeiss (6 mentions)
Formaldehyde, by Merck Group (5 mentions)
Millicell ez slide, by Merck Group (5 mentions)
Cyanine3 (cy3), by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (4 mentions)
Lsm800 confocal microscope, by Zeiss (4 mentions)
Ab1793, by Abcam (3 mentions)
Dylight 647 and 488, by Thermo Fisher Scientific (3 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Cm3050, by Leica camera (2 mentions)
Tissue tek oct compound, by Sakura Finetek (2 mentions)
Phalloidin 568, by Thermo Fisher Scientific (2 mentions)
109 protocols using prolong diamond antifade reagent
1
NEAT1 RNA Localization by Fluorescence In Situ Hybridization
2
Visualizing BCR Signaling Activation
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Primary murine B cells (105 in 0.1 ml imaging medium) from C57BL/6J or MD4 mice that had been pre-treated with CK-689 or CK-666 were added to COS-7 APCs expressing either the single-chain anti-Ig surrogate Ag or mHEL-HaloTag that was labeled with HaloTag tetramethylrhodamine ligand (Promega, #G8251). After the indicated times at 37°C, the cells were fixed with 4% PFA for 10 min at room temperature. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 3 min and blocked with 2% BSA in PBS for 30 min, both at room temperature. Primary antibodies that were diluted in blocking buffer (2% BSA in PBS) were added for 1 hr at room temperature, or overnight at 4°C for anti-pCD79. The primary antibodies, all from Cell Signaling Technologies, were: pZap70(Y319)/pSyk(Y352) (#2701; 1:200), pCD79a(Y182) (#5173; 1:400), and pCD19(Y531) (#3571; 1:200). The coverslips were then incubated for 30 min at room temperature with a mixture of Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, #A-11008; 1:400) secondary antibody, fluorophore-conjugated phalloidin to visualize F-actin, and Alexa Fluor 647 goat anti-rat IgG (Invitrogen, #A-21247; 1:400) to detect the rat IgG1 portion of the surrogate Ag and thereby visualize Ag clusters. Coverslips were mounted onto slides using ProLong Diamond anti-fade reagent (Thermo Fisher, #P36965).
3
Retinal Tissue Preparation for Microscopy
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Mice treated with AAV-PHP.B were anesthetized and fixed as described above. Their eyes were removed and frozen in Tissue-Tek O.C.T. compound (Sakura Finetek Japan, Tokyo, Japan). The 16-μm-thick retinal slices were prepared using a cryostat (CM3050S, Leica) and mounted on glass slides with Prolong Diamond Antifade Reagent (Thermo Fisher Scientific).
4
Immunofluorescence Staining Protocol for Parasites
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Parasites were fixed with freshly made 4% paraformaldehyde (Electron microscopy sciences, Hatfield, PA; Cat# 15714) in 1xPBS (ThermoFisher; Cat# 18912–014) for 15 minutes at RT. Cells were washed three times in 1xPBS and permeabilized in 0.25% TX-100 diluted in 1xPBS for 10 minutes at room temperature before washing three times in 1xPBS. Cells were blocked in 2% BSA-1XPBS for 15 minutes before antibody incubations. All antibodies were diluted in 0.5% BSA-1xPBS at the concentrations indicated in S4 Table . DNA was stained with 10μM DAPI diluted in 1xPBS for 10 minutes and then washed three times in 1xPBS. Cells in mattek dishes were either imaged immediately or stored in 1xPBS at 4°C. Coverslips were mounted onto slides using either Prolong Gold anti-fade reagent (ThermoFisher; Cat # P36930) or Prolong Diamond anti-fade reagent (ThermoFisher; Cat #P36965) and allowed to dry overnight before imaging.
5
Immunohistochemistry and Immunofluorescence Protocols
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Immunohistochemistry (IHC) and immunofluorescence (IF) were performed according to previously described procedures [23 (link),50 (link)]. Eyeballs were fixed with fixative solutions (10% formalin and 4% PFA), washed, and stored in PBS until use. The fixed specimens were embedded with paraffin or cryopreserved with OCT compounds for sectioning. The sections were permeabilized by incubation in 0.05% Triton X-100 for 10 min and blocked with 2.5% normal goat serum for 1 h at room temperature (RT). The samples were incubated with primary antibodies (Table 1 ) and appropriate secondary antibodies coagulated with chemical enzymes or fluorescent dye. For IHC, horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000; Bio-Rad, Hercules, CA, USA) was applied and incubated at room temperature for 1 h. The staining signals were visualized by incubation with DAB substrate (34002; Thermo Fisher, Waltham, MA, USA) for 7 min. For IF staining, the signals were visualized by the secondary antibodies coagulated with Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, or Cyanine5. The cell nuclei were visualized by incubation with 4′,6-diamidino-2-phenylindole (DAPI). The slides were mounted with ProLong Diamond antifade reagent (Thermo Fisher, Waltham, MA, USA). The images were taken and analyzed with light, fluorescent, or confocal microscopy.
6
STED Microscopy Protein Detection
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Free-floating sections were first permeabilized with 0.25% TritonTM-X100 (TX, Sigma-Aldrich) and incubated in blocking solution [1X PBS containing 0.25% TX, 4% normal goat serum (NGS, Thermo Fisher Scientific, Waltham, Massachusetts, United States)] for 30 min at room temperature, to permeabilize membranes and saturate non-specific sites. Co-detection of two proteins was carried out by incubation in a mixture of primary antibodies recognizing each of the proteins of interest, diluted in a solution of 1X PBS-0.25% TX–1% NGS. After incubation overnight at room temperature with stirring, sections were washed in 1X PBS-0.25% TX, then incubated for 2 h with the secondary antibodies coupled to different fluorophores (Alexa-594 and Abberior-Star 635P; dilution: 1: 100 in 1X PBS-0.25% TX). All fluorophores used are suitable for confocal and STED microscopy. They were washed again in 1X PBS-0.25% TX, rinsed in 1X PBS, then mounted on slides with ProLong Gold or ProLong Diamond Antifade Reagent (Thermo Fisher Scientific). Sections were then observed and acquired in a STED super resolution microscopy (LEICA SP8—STED 3X microscope) equipped with a 775 nm depletion laser, respectively (Leica Microsystems, Wetzlar, Germany).
7
Cytotoxic T Cell Degranulation Assay
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CTLs were washed and allowed to adhere for 12 minutes onto hydrophobic mutliwell slides (Hendles-Essex) in FCS-free IMDM. For conjugation assays, blue P815 target cells were coated with anti-CD3 (UCHT1), plated on multiwall slides in serum free media and CTLs were added for 10-15min. Cells were fixed with 4% paraformaldehyde (15710-S, Electron Microscopy Systems, USA) and incubated in quenching solution (50 mM ammonium chloride in 1X PBS) (15 min at RT) and rinsed twice in PBS. Permeabilization was done with 0.2% Saponin in PBS (5 min, RT) followed by washing and blocking for 30min in blocking buffer (1xPB, 1%BSA, 0.2% saponin). Cells were stained with anti-LAMP1 (H4A3, hybridoma supernatant), anti-RAB27A (abcam, UK) for 1 hour, followed by fluorophore-conjugated secondary antibodies (donkey anti-mouse 488 and goat anti-rabbit 647, Invitrogen, UK) (1 hour at RT) together with phalloidin 568 (Invitrogen, UK). Nuclei were stained for 5 minutes at RT with Hoechst (Thermo Fisher, UK), and samples were mounted in ProLong Diamond Antifade Reagent (Thermo Fisher, UK).
8
Enhancing GFP Signal in Fixed Tissue
9
Quantification of Virus RNA-Protein Colocalization
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Cells were grown on glass coverslips in 24 wells plates. After infection at high MOI for the indicated times, cells were fixed with PBS-formaldehyde 4% (v/v) for 10 min, washed with PBS and permeabilized with PBS-BSA 1% (w/v)-Triton X-100 0.25% (v/v) for 10 min. Cells were incubated for 1 h in PBS-BSA 1% (w/v) with the appropriate primary antibodies, rinsed with PBS then incubated for 30 min with the appropriate Alexa Fluor-conjugated secondary antibodies and with Hoechst 33342 (1 μg/ml). Coverslips were rinsed in PBS, then mounted in ProLong diamond antifade reagent (Thermofisher). Cells were examined by confocal microscopy under a WLL Leica SP8 microscope except indicated otherwise. Representative pictures were taken.
Colocalization analysis were performed using the open-source software Icy (http://icy.bioimageanalysis.org/ ) [58 (link)]. We used Spot Detector plugin to automatically detect objects corresponding to RNP and vesicles (Rab11a or EEA1) and then Colocalization Studio plugin method: object based and statistical colocalization, SODA [35 (link)], to quantify the number of RNP at a distance less than 270 nm from the cellular vesicles.
Colocalization analysis were performed using the open-source software Icy (
10
Corneal Mucin Expression Analysis
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Following two weeks of daily TcES or TpES, mice were sacrificed by carbon dioxide inhalation with secondary cervical dislocation. The eyes were immediately enucleated and fixed with 4% paraformaldehyde (VVR Life Science, Radnor, PA, USA) at 4 °C overnight. The corneas were then carefully dissected under the microscope and subjected to blocking and permeabilization in Triton X-100 (0.5%) with 5% normal donkey serum overnight at 4 °C. The samples were then incubated with a primary antibody to MUC4 (1:100; AB1793, Abcam, Cambridge, MA, USA) for 24 h at 4 °C and washed with PBST 3 times for 30 min. After PBST washing, corneal specimens were visualized by donkey anti-mouse IgG (H + L) and Cyanine3 (A10521, 1:1000; Fisher Scientific, Waltham, MA, USA). Specimens were mounted on slides under the microscope, and 4 relaxing cuts were made to flatten the tissue and covered with a ProLong Diamond antifade reagent (Thermo Fisher Scientific, Waltham, MA, USA). The central corneal area was then imaged by confocal microscopy.
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