4×104 cells per well were cultured in 24-well plates. After adherence, cells were fixed by 4% paraformaldehyde for 15 min at room temperature. After washed with PBS, cells were blocked in 5% normal goat serum with 0.3% Triton-X100 for 30 min. Cells were incubated with a mixture of primary antibodies for overnight at 4℃ after washed with PBS. The primary antibodies used were as follows: mouse monoclonal anti-DTX3 (1:100, cat. no. sc-376439, Santa Cruz Biotechnology, Inc) and rabbit polyclonal anti-XRCC5 (1:200, cat. no. 16389-1-AP, Proteintech Group Inc). Then cells were washed with PBS 3 times for 5 min and incubated with a mixture of secondary antibodies, goat anti-mouse IgG Dylight 594 (1:100) and goat anti-rabbit IgG Dylight 488 (1:100), for 1 h. For nuclei staining, cells were counterstained with DAPI (cat. no. C0065, Solarbio Life Sciences) for 5 min and mounted with Antifading Mounting Medium (cat. no. S2100, Solarbio Life Sciences) for confocal image acquisition (400× magnification).
Antifading mounting medium
Manufactured by Solarbio
Sourced in China
Antifading mounting medium is a product designed to preserve fluorescent signals in microscopy samples. It prevents the fading or loss of fluorescence over time, allowing for longer observation and imaging of labeled specimens.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Solarbio (2 mentions)
Anti xrcc5, by Santa Cruz Biotechnology (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
Triton x 100, by Beyotime (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Neun antibody, by Proteintech (1 mentions)
Triquick reagent, by Solarbio (1 mentions)
Staurosporine, by AbMole (1 mentions)
Confocal fluorescence microscopy, by Leica (1 mentions)
β catenin antibody, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Ab184330, by Abcam (1 mentions)
Sc 6259, by Santa Cruz Biotechnology (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Ab81299, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
10 protocols using antifading mounting medium
1
Immunofluorescence Assay of DTX3 and XRCC5
2
Immunofluorescence Imaging of DLK1 and NCOR1
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The cells were seeded on coverglass placed in six-well plates. Cells then were fixed in cold methanol at 4°C for 10 min and permeabilized in 0.5% Triton X-100 PBS solution 4°C for 10 min. After several washings by PBST, cells were blocked in 5% skim milk PBST 1 h at room temperature. For single label, cells were incubated with rabbit polyclonal anti-DLK1 antibody at 4°C overnight. For double label, cells were incubated with rabbit polyclonal anti-DLK1 antibody and mouse monoclonal anti-NCOR1 antibody together at 4°C overnight. They then were washed three times in PBST and incubated with goat anti-rabbit Alexa Fluor® 488–conjugated antibody (DLK1) for 1 h and/or with goat anti-mouse Alexa Fluor® 555–conjugated antibody (NCOR1) for 1 h. Finally, after washing in PBST, cells were incubated with DAPI to visualize the nuclei. The coverglass were mounted face down on an air-dried glass slide using antifading mounting medium (Solarbio, Beijing). The slides were viewed on Zeiss Laser-scanning confocal microscope equipped with Zeiss image processing software (TCL SP8).
3
Goblet Cell Enumeration in Colon Tissue
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The proximal colons were fixed in paraformaldehyde (4% in PBS, v/v) and embedded in paraffin. For H&E staining, colon sections were cut and stained with hematoxylin and eosin. Three animals per group were used for the assessment of goblet cell numbers. The number of goblet cells on each villus was counted.
For immunofluorescence staining, sections were exposed to Claudin-1 antibodies (Abcam, Ab15098, USA) at 4°C for 10 h, and incubated with biotinylated antibodies. After DAPI staining, the sections were sealed with antifading mounting medium (Solarbio, Beijing, China). Stained sections were observed using an inverted fluorescence microscope (Olympus, Tokyo, Japan).
For immunofluorescence staining, sections were exposed to Claudin-1 antibodies (Abcam, Ab15098, USA) at 4°C for 10 h, and incubated with biotinylated antibodies. After DAPI staining, the sections were sealed with antifading mounting medium (Solarbio, Beijing, China). Stained sections were observed using an inverted fluorescence microscope (Olympus, Tokyo, Japan).
4
TUNEL Assay for Apoptosis Detection
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Tissue sections with a thickness of 5 µm were dewaxed with xylene (Aladdin Reagent) and rehydrated with gradient ethanol. The 0.1% TritonX-100 (Beyotime) solution was used
to permeabilize tissue sections, which were repaired by citric acid-sodium citrate solution at a high temperature for 10 min. After being labeled by TUNEL solution (In Situ Cell Death
Detection Kit, Roche, Basel, Switzerland), tissue sections were blocked with 1% BSA solution and incubated with primary antibody (1:50) at 4°C overnight and secondary antibody (1:200) at
room temperature for 60 min, followed by 4′,6-diamidino-2-phenylindole (DAPI) (Aladdin Reagent) counterstaining. Tissue sections were mounted with Antifading Mounting Medium (Solarbio) and
imaged using a microscope (BX53, OLYMPUS). Neuronal nuclei antigen (NeuN) antibody was purchased from Proteintech (Hubei, China). Goat anti-rabbit-Fluorescein Isothiocyanate (FITC) IgG was
purchased from Abcam (Cambridge, UK).
to permeabilize tissue sections, which were repaired by citric acid-sodium citrate solution at a high temperature for 10 min. After being labeled by TUNEL solution (In Situ Cell Death
Detection Kit, Roche, Basel, Switzerland), tissue sections were blocked with 1% BSA solution and incubated with primary antibody (1:50) at 4°C overnight and secondary antibody (1:200) at
room temperature for 60 min, followed by 4′,6-diamidino-2-phenylindole (DAPI) (Aladdin Reagent) counterstaining. Tissue sections were mounted with Antifading Mounting Medium (Solarbio) and
imaged using a microscope (BX53, OLYMPUS). Neuronal nuclei antigen (NeuN) antibody was purchased from Proteintech (Hubei, China). Goat anti-rabbit-Fluorescein Isothiocyanate (FITC) IgG was
purchased from Abcam (Cambridge, UK).
5
Molecular Probes for Neural Analysis
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Staurosporine (Cat# M2066) and N-methyl-d -aspartic acid (NMDA, Cat# M2884) were acquired from AbMole (Beijing, China); 4’,6-diamidino-2-phenylindole (DAPI, Cat# C0065), antifading mounting medium (Cat# S2100), and Triquick Reagent (Trizol Substitute, Cat# R1100) were purchased from Solarbio (Beijing, China); Cholera Toxin Subunit B (CTB) conjugated Alexa Fluor 488 (Cat# C34775) was obtained from Thermo Fisher Scientific; and murine rCXCL2 (Cat# 250–15) was acquired from PeproTech (Rocky Hill, NJ, USA).
6
Immunofluorescence Assay for NLRP3 and β-Catenin in Placental Tissues
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For placental tissues, paraffin-embedded sections were subjected to a standard deparaffinization and rehydration process and incubated in citric acid for antigen retrieval. After that, the sections were blocked with 1% bovine serum albumin (BSA) at 37°C for 30 min and then incubated with primary NLRP3 antibody (1:100, Proteintech, Wuhan, China) or β-Catenin antibody (1:500, ab32572, Abcam) at 4°C overnight. As a secondary antibody, fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (1:500, Proteintech, Wuhan, China) was added for incubation at room temperature for 50 min. Nuclei were stained with DAPI (Solarbio, Beijing, China) under the conditions of protection from light for 10 min, and then the sections were sealed with antifading mounting medium (Solarbio, Beijing, China). Immunofluorescent images were visualized with confocal fluorescence microscopy (Leica, Germany), and the pixel intensity of the immunoreactive signal was measured using ImageJ (NIH).
7
Immunofluorescence Staining of Uterine Tissue
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Immunofluorescence was performed as previously described [62 (link)]. In brief, frozen uterine sections (10 μm) were cut and fixed in freshly prepared 4% paraformaldehyde in PBS for 30 min. After being permeabilized with 0.1% Triton X-100 in PBS for 15 min and blocked with 10% horse serum (Zhongshan Golden Bridge, Beijing, China) at 37 °C for 1 h, sections were incubated with primary antibody for anti-laminin A5 (ab184330, Abcam, MA, USA), anti-laminin B2 (05-206, Sigma-Aldrich), anti-laminin C1 (ab233398, Abcam), anti-CK18 (sc-6259, Santa Cruz Biotechnology, Dallas, TX, USA), or anti-IGFBP1 (sc-55474, Santa Cruz Biotechnology) overnight at 4 °C. After washing with PBS, sections were incubated with 488- or 594-conjugated second antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at 37 °C for 30 min. The section was then counterstained with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Sigma-Aldrich), and mounted with an antifading mounting medium (Solarbio Life Sciences, Beijing, China).
8
X-ray Induced DNA Damage Visualization
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Cells were irradiated with a dose of 4 Gy x‐ray. The distance to the radiation source was 100 mm. After irradiation, cells were fixed using 4% paraformaldehyde (Solarbio) for 10 min at room temperature. Then, fixed cells were permeated by 0.2% Triton X‐100 for 10 min, followed by blocking using 5% BSA for 1 h. Cells were incubated with primary antibody against γ‐H2A.X (ab81299, Abcam) overnight at 4℃. After wash, the cells were incubated with DyLight 649 goat anti‐rabbit IgG (H+L) secondary antibody (GAR6492, Multisciences) for 1 h at room temperature and then stained with DAPI containing antifading mounting medium (Solarbio).
9
Immunofluorescence Staining of PSMD11 in MEFs
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MEFs were fixed in 4% buffered formalin for 20 min, after washing twice with PBS, it was blocked for 1 h PBS containing 3% (w/v) bovine serum albumin (BSA), 1% (w/v) Saponin and 1% (v/v) Triton X-100, then incubated with rabbit anti-PSMD11 antibody (1:100, 14,786–1-AP, Proteintech Group, Rosemont, USA) for 24 h at 4 °C in the dark followed by FITC conjugated goat anti-rabbit antibodies (#0311, Zhongshan Goldenbridge Biotechnology Co., LTD) for 1 h at room temperature. Nuclei were counterstained with DAPI (1:1000, Invitrogen) for 5 min at room temperature. After three rinses in PBS, slides were mounted with antifading Mounting Medium (Solarbio, Beijing, China) and imaged with fluorescent microscopy with appropriate excitation wavelengths.
10
Visualizing RNA Dynamics in Cell Lines
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Hela cells and HEK293T cells were cultured in 24-well plates with poly-d -lysine coverslips, and then these cells were co-transfected with 200 ng VN-dEcCas6-VC-GK and 800 ng Actin-16 × CBS-GK or hTERC-16 × CBS-GK. Single molecule inexpensive fluorescence in situ hybridization (smiFISH) was employed for RNA imaging (11 (link)). The sequences of primary probes and FLAP probes were listed in Supplementary Table S1 . The smiFISH was performed according to the previously reported method. Briefly, cells were fixed with 4% PFA for 15 min at 37°C and washed with 1 × PBS (pH7.4) for 5 min three times, followed by permeabilization with 0.5% Triton X-100 for 15 min, and these cells were rewashed with 1 × PBS (pH7.4) for 5 min three times. Cells were incubated in hybridization solution at 37°C for 12 h. After hybridization, the samples were washed with 15% formamide (in 1 × SSC) at 37°C for 30 min three times, stained with 0.1 μg/ml DAPI at room temperature for 10 min, and mounted in antifading mounting medium (Solarbio).
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