GC cells transfected with pRK5-FLAG-USP13 and pcDNA3-Myc-cyclin D1 plasmids for 48 h were seeded on a glass coverslip. The cells were fixed for 25 min in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (in PBS) for 10 min, followed by blocking with 5% BSA for 30 min at room temperature. Then the cells were incubated with primary antibodies against Flag (Sigma-Aldrich, 1:500) and Myc (ABclonal, AE070, 1:250) at 4 °C overnight. After three PBS washes, the cells were incubated with CoraLite594 (Proteintech, SA00013–3, 1:500) and CoraLite488 (Proteintech, SA00013–2, 1:500) conjugated secondary antibodies for 2 h at room temperature in the dark. Cells were washed three times with PBS in the dark, stained with DAPI and mounted in antifade mounting reagent (Beyotime, P0126). The immunofluorescent staining was observed with a confocal microscope (ZEISS Axio Scope. A1, Germany).
Coralite488
Manufactured by Proteintech
Sourced in United States
CoraLite488 is a fluorescent dye that emits light in the green spectrum when excited. It is commonly used in immunofluorescence and other fluorescence-based techniques to label and visualize target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Coralite594, by Proteintech (4 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Antifade mounting reagent, by Beyotime (1 mentions)
Axio scope a1, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Ab53707, by Abcam (1 mentions)
Ab108297, by Abcam (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Cas block solution, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Ab71973, by Abcam (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Anti phlpp2, by Abcam (1 mentions)
Anti phospho akt s473, by Cell Signaling Technology (1 mentions)
Sa00013 1, by Proteintech (1 mentions)
Sa00013 4, by Proteintech (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Anti flag, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Er tracker red, by Beyotime (1 mentions)
15 protocols using coralite488
1
Visualizing USP13 and Cyclin D1 Interaction
2
Immunofluorescence Staining of K562 Cells
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K562 cells were centrifuged on glass coverslips, fixed with 4% paraformaldehyde in PBS for 20 min, and subsequently permeabilized with 0.1% Triton X-100 for 10 min. After blocking with 1% BSA for 1 h at room temperature , cells were incubated with primary antibody overnight at 4 °C, followed by secondary antibody conjugated with CoraLite488 (SA00013-1, Pro-teintech, IL, USA) for 1 h at room temperature. After three washes with PBS, cells were mounted with Aqueous Mounting Medium containing 4',6-diamidino-2-phenylindole (DAPI, Beyotime, C1005, Shanghai, China) and visualized with a confocal microscope.
3
Immunofluorescence Staining of Cultured Cells
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Cultured cells were washed once with PBS and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 10 min at RT. Samples were washed twice with PBS, and 0.1% Triton-X solution was added to promote membrane permeability by the antibody against cytokeratin 17. The samples were washed three times and then treated with CAS-Block solution (008120, Thermo Fisher Scientific, MA, U.S.A.) for 30 min at RT. Primary antibodies were diluted with CAS-Block solution and then applied to the samples, and the treated samples were incubated at 4 °C overnight. We used the following antibodies at a 1:100 dilution: anti-cytokeratin 17 antibody (ab53707, Abcam) and anti-CD99 antibody [EPR3096] (ab108297, Abcam), a 1:50 dilution: EZH2 antibody (21,800–1-AP, proteintech). After rinsing four times, for 15 min each time, in PBS, the samples were incubated at room temperature for 3 h with fluorescent secondary antibodies (Alexa-488, Invitrogen, CA, U.S.A. or CoraLite488, proteintech, IL, U.S.A.). The samples were observed under a fluorescence microscope after four final rinses, each for 15 min each time, in PBS.
4
Subcellular Localization of ZDHHC22
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Cells were seeded on coverslips and then transfected with pCMV6-Entry, pCMV6-ZDHHC22, or pCMV6-ZDHHC22-(C111A). After 48 hours, cells were fixed with 4% paraformaldehyde for 20 min and then permeabilized with 0.5% Triton X-100 for 10 min. After blocking with blocking buffer, cells were incubated with primary antibodies overnight at 4°C and then incubated with secondary antibodies for 1 hour at 37°C. DAPI (Roche, Palo Alto, CA, USA) was used for DNA counterstaining. Photomicrographs were acquired with a confocal laser scanning microscope (Leica, Hilden, Germany). The following antibodies were used for immunofluorescence: anti-Flag (1:400, #14793, Cell Signaling Technology), anti-mTOR (1:200, #2983, Cell Signaling Technology), anti-phospho-AKT(S473) (1:200, #4060T, Cell Signaling Technology), anti-PHLPP2 (1:40, ab71973, Abcam), ER-Tracker Red (1:2000, C1041, Beyotime), CoraLite488 (1:200, SA00013-1, Proteintech), and CoraLite594 (1:200, SA00013-4, Proteintech).
5
Immunofluorescence Staining of Cells
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Cells were plated in a glass-bottom cell culture dish (BS-15-GJM, Biosharp, Hefei, China) and fixed in 4% paraformaldehyde at room temperature for 15 min when the cell confluence reached 60%–80%, and then the cells were permeabilized with 0.3% Triton X-100 for 20 min. Next, the cells were blocked with PBS containing 5% goat serum for 1 h at room temperature, followed by incubation with the indicated primary antibodies (Supplementary Table S2 ) overnight at 4 °C. After washing with PBS, the cells were incubated with goat anti-rabbit IgG conjugated with CoraLite488 (1:500) (SA00013-2, Proteintech, Wuhan, China) in the dark for an hour and then incubated with antifade mounting medium with DAPI (P0131, Beyotime, Shanghai, China) for 10 min. The signals from targeted proteins were visualized using a confocal laser scanning microscope (LSM 800, Zeiss, Oberkochen, Germany).
6
Immunofluorescence Staining of Cells on Hydrogels
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Cells cultured on hydrogel with different stiffness were fixed in 4% paraformaldehyde (P1110, Solarbio) for 30 min and permeabilized with 0.2% Triton X-100 (T8200, Solarbio) for 20 min. The cells were then blocked with 10% goat serum (AR1009, Boster) for 1 h and incubated with anti-Ki67 antibody (27309-1-AP, Proteintech) or anti-YAP antibody (13,584–1-AP, Proteintech) overnight at 4 ℃. Subsequently, the cells were incubated with CoraLite488-conjugated secondary antibody (SA00013-2, Proteintech) or rhodamine-conjugated secondary antibody (SA00007-2, Proteintech) for 1.5 h in the dark. F-actin was stained with rhodamine-phalloidin (CA1610, Solarbio). Nuclei were counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (AR1176, Boster), and images were captured by fluorescence microscopy (Olympus, IX73). For tissue sections, after deparaffinization and dehydration, the remaining protocol was the same as that for cells.
7
Immunofluorescent Staining of Mouse Tumor Cells
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Fresh mouse tumor mass was isolated and embedded in OCT blocks frozen to prepare 6 mm thick frozen sections. Cells were inoculated on coverslips at the bottom of a 48-well plate (2–4 × 104 cells/well). Cells and tissues were fixed in 4% paraformaldehyde for 30 min at room temperature (RT) and then permeabilized in 0.5% Triton X-100 solution for 10 min. Non-specific sites were blocked with 5% bovine serum albumin for 30 min at RT and incubated with primary antibody overnight at 4 °C. After washing with PBS, CoraLite488 or 594 secondary antibodies (Proteintech) were incubated for 1 h at RT, then stained with Hoechst (Solarbio) for 10 min at RT. Coverslips with the anti-fading solution were mounted on glass slides and observed under a Zeiss LSM 880 confocal microscope.
8
Immunofluorescence Staining of PDLSCs
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PDLSCs were treated under different conditions on the cell climbing sheets for 1 day. Fixed by paraformaldehyde, 1000 μL blocking buffer (0.2% Triton-100 and 5% donkey serum) was added onto each sheet. Then, the cells were incubated with the primary antibody (VEGF-a or LC3B) at 4°C overnight. After rewarming to room temperature, the cells were dealt with the secondary antibody, DyLight680 (Invitrogen, CA, USA) for 1 hour. DAPI and phalloidin (CoraLite488, Proteintech, Wuhan, China) were used to stain cell nucleus and cytoskeleton. Images of cells were observed and captured under Olympus SpinSR confocal laser microscope (Olympus, Tokyo, Japan).
9
Immunohistochemical Analysis of ESCC Tissues
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The expression of protein in 152 ESCC tissues was detected using an immunoperoxidase method. IHC staining was performed according to standard protocols as previously described [24 (link)]. The slides were incubated in primary antibody (LTBP1(1:1000, #26855; Proteintech), FN1 (1:2000, #66042; Proteintech), E-Cadherin (1:200, #3195; Cell Signaling) and TGFβ1(1:60, #ab92486, Abcam) and followed by treatment with secondary antibody and a DAB staining kit (Gene Tech #GK500705). IHC intensity for each tissue section was evaluated independently by 2 experienced pathologists who were blinded to patients’ clinical data.
Cells were incubated in primary antibodies α-SMA (1:100, #14395; Proteintech) and FN1 (1:100, #66042; Proteintech). The cells were washed with PBS for three times and followed by treatment with CoraLite488 and CoraLite594 labeled IgG (Proteintech) for an hour. After washing the cells for four times, cells were stained by DAPI (Sigma).
Cells were incubated in primary antibodies α-SMA (1:100, #14395; Proteintech) and FN1 (1:100, #66042; Proteintech). The cells were washed with PBS for three times and followed by treatment with CoraLite488 and CoraLite594 labeled IgG (Proteintech) for an hour. After washing the cells for four times, cells were stained by DAPI (Sigma).
10
Immunofluorescence Staining of Cellular Markers
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NP cells attached to slides were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, permeabilized with 0.5% Triton X-100 for 15 min, blocked with 2% bovine serum albumin (BSA) for 30 min, and then incubated overnight at 4°C with primary antibodies against p16 (1 : 100, Proteintech), cleaved caspase 3 (1 : 100, CST), FAM134B (1 : 100, Proteintech), and LC3 (1 : 100; Abconal, Wuhan, China). After washed three times with TBST, cells were incubated with CoraLite488 or CoraLite594 conjugated goat anti-rabbit/mouse IgG antibody (1 : 100, Proteintech) for 1 h and labeled with diamidino-2-phenylindole (DAPI; Beyotime) for 5 min and then observed images using a fluorescence microscope (Olympus).
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