SMSCs were seeded on glass coverslips in 24-well plates and treated as the experiment group processing. After another 24 hours, the chondrogenic induction cells were fixed and subsequently treated with 0.3% Triton X-100 (MP Biomedicals, USA). After washed with PBS, the cells were then incubated in 5% BSA. Then, the cells were incubated with microtubule-associated protein light chain 3 (LC3) rabbit monoclonal antibody (1 : 100; Abcam, USA) overnight at 4°C. The negative control group was treated with 1% BSA instead of the primary antibody. The FITC-conjugated goat antirabbit IgG (1 : 100; Proteintech, USA) was added for 1 hour and then incubated with DAPI staining solution (Leagene, China) for 15 min. The coverslip was dried and sealed with antifluorescence quenching agent (Beyotime, China). The cells were observed under laser confocal microscopy (FV10i, Olympus, Japan).
Fitc conjugated goat anti rabbit igg
Manufactured by Proteintech
Sourced in United States, China, Japan
FITC-conjugated goat anti-rabbit IgG is a secondary antibody that binds to rabbit primary antibodies. The FITC (fluorescein isothiocyanate) label allows for fluorescent detection of the target rabbit antibody.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Cy3 conjugated goat anti rabbit igg, by Proteintech (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Fv1200 microscope, by Olympus (2 mentions)
Triton x 100, by MP Biomedicals (1 mentions)
Anti fluorescence quenching agent, by Beyotime (1 mentions)
Fv10i, by Olympus (1 mentions)
Dapi staining solution, by Leagene (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated goat anti mouse igg, by Proteintech (1 mentions)
Immunol staining blocking buffer, by Beyotime (1 mentions)
Lamin b1, by Cell Signaling Technology (1 mentions)
Immunol staining fix solution, by Beyotime (1 mentions)
Fv1000, by Olympus (1 mentions)
Normal goat serum (ngs), by Solarbio (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Anti β catenin, by Proteintech (1 mentions)
Pam3csk4, by InvivoGen (1 mentions)
24 well culture plate, by Corning (1 mentions)
16 protocols using fitc conjugated goat anti rabbit igg
1
Chondrogenic Induction Cell Autophagy
2
SARS-CoV-2 Propagation and Antibody Detection
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Vero-E6 cells (ATCC CRL-1686) and Caco2 cells (ATCC HTB-37) were kindly provided by Prof. Zheng-Li, Shi (Wuhan institute of Virology, CAS). BHK-21 cells were kindly provided by Prof. Han-Zhong, Wang (Wuhan institute of Virology, CAS). For all cell culture procedures, the cells were cultivated in Dulbecco’s modified Eagle Medium (DMEM; Invitrogen, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Gibco), and penicillin (100 U/ml)-streptomycin (100 μg/ml) at 37°C with 5% CO2. SARS-CoV-2 (WIV-04) (3) was propagated in Vero E6 cells and stored in aliquots at −80°C for experiments. All the experiments using the live SARS-CoV-2 virus were performed in a BSL-3 laboratory at Wuhan Institute of Virology according to standard BSL-3 guidelines. The antibody against the NP protein of RP3-CoVwas kindly provided by Prof. Zheng-Li, Shi (Wuhan institute of Virology, CAS), which is cross-reactive with the NP protein of SARS-CoV-2. FITC-conjugated goat anti-rabbit IgG was purchased from Protein Tech Group. Berbamine hydrochloride, liensinine, isoliensinine, tetrandrine, fangchinoline and cepharanthine were purchased from Weikeqi Biotech (Sichuan, China).
3
Immunofluorescence Assay of Cell Cycle Markers
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HK-2 cells were plated in 12-well plates with 20 mg/mL BSA for 72 h. After 72 h of treatment with BSA or si-p21 transfection, the cells were fixed with an immunol staining fix solution (Beyotime) for 15 min and blocked with an immunol staining blocking buffer (Beyotime) for 30 min. Then, after being rinsed with PBS for three times (5 min each), the cells were incubated with primary antibodies against p-PH3Ser10 (1:500; Cell Signaling Technology, Beverly, USA) and LaminB1 (1:50; Cell Signaling Technology) at 4°C overnight. After three times wash with PBS, the cells were incubated with FITC-conjugated goat anti-rabbit IgG (1:100; ProteinTech, Rosemont, USA) or FITC-conjugated goat anti-mouse IgG (1:100; ProteinTech) secondary antibody in the dark for 1 h. Finally, the nuclei were counterstained with DAPI. The slides were visualized for immunofluorescence and FISH with a fluorescence microscope at a magnification of ×400. The images were analyzed using ImageJ software.
4
Immunofluorescence Localization of BbAFP1 in P. pastoris
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The immunofluorescence localization in P. pastoris was performed as previously described (Wang et al., 2018 ). Briefly, P. pastoris strains induced with methanol for 4 days were incubated with normal goat serum (final concentration of 5%, Solarbio, Beijing, China) at 4 °C overnight. After washing three times with PBS (10 mm , pH 6.0), the samples were incubated with anti‐BbAFP1 rabbit antibody (1:1000 (v/v) dilution) for 3 h and then incubated with FITC‐conjugated goat anti‐rabbit IgG (1:500 (v/v) dilution, Proteintech, Chicago, Illinois) for 2 h. The green fluorescence on the cell surfaces of P. pastoris was visualized using confocal microscopy (FV1000; Olympus, Tokyo, Japan).
5
Tachyzoite Proliferation Assay in HFF Cells
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The effect of the test compounds on tachyzoite proliferation was studied using HFF. Freshly released tachyzoites (1×104 tachyzoites/well) were inoculated in confluent HFF on 12-well plates for 30 min to allow cell invasion, followed by extensive washing and further incubation in DMEM with 2% FBS and 0.1 mM sterculic acid or 1 mM methyl esters for 18 hr. Subsequently, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100. The intracelullar parasites were incubated with anti-T. gondii rabbit sera (1:50) and then FITC-conjugated goat anti-rabbit IgG (1:50, Proteintech Group Inc., Chicago, Illinois, USA) for 1 hr at 37˚C for each antibody incubation step. Tachyzoites labelled with green fluorescence were observed under an inverted microscope (Olympus), and intracellular parasites at different proliferation stages (i.e. 1, 2, 4, or 8 tachyzoites) were counted from 100 parasitophorous vacuoles in 3 separate wells per experiment.
6
Immunofluorescent Labeling of β-Catenin
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Immunofluorescent labeling was performed on cell-bearing coverslips using the method described previously [12 (link)]. The CAL-62 cells on cell-bearing coverslips were treated with 100 μM ART or 0.2% DMSO for 48 h. The working concentration of rabbit anti-β-catenin used for immunofluorescence staining was 1:500. Briefly, the cell-bearing coverslips were washed in phosphate-buffered saline (PBS, pH 7.4), incubated in 3% H2O2 for 10–15 min, and further incubated with anti-β-catenin (1:500; Proteintech) overnight in a humid chamber at 4 °C. Lastly, the coverslips were co-incubated with FITC-conjugated goat anti-rabbit IgG (1:100; Proteintech) at 37 °C for 1 h in the dark, sealed with anti-fluorescence quenching sealing liquid (including DAPI; Beyotime Biotech), and imaged using a fluorescence microscope (Ni-U, Nikon, Tokyo, Japan).
7
Localization of NF-κB/p65 in Macrophages
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To determine the localization of NF-κB/p65, 5 × 105 PMs were seeded in 24-well culture plates (Corning Incorporated, Corning, NY, United States) with sterile glass slides, and the cells were treated with rNc14-3-3 (50 μg/ml) or Pam3CSK4 (TLR2/TLR1 Agonist, InvivoGen, United States, 10 ug/ml) at 37°C. After 30 min, the cells were washed twice with sterile PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton X-100 in PBS for 10 min, and washed. Then, the cells were incubated with a rabbit anti-phospho-NF-κB/p65 antibody (Santa Cruz, CA, United States) at a 1:100 dilution at 4°C overnight. The cells were washed and incubated with FITC-conjugated goat anti-rabbit IgG (Proteintech, Wuhan, China) for 1 h at RT. The cells were washed, and the coverslips were stained with DAPI before analysis on a Zeiss LSM 710 confocal microscope equipped with a 633, 1.4-NA, oil-immersion objective (Carl Zeiss).
8
Evaluating SARS-CoV-2 receptor expression
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HeLa cells transiently expressing APN, ACE2 or DPP4 were prepared using Lipofectamine 2000 (Thermo Fisher Scientific) in a 96-well plate, with mock-transfected cells as controls. SADS-CoV grown in Vero cells was used to infect HeLa cells transiently expressing APN, ACE2 or DPP4. The inoculum was removed after 1 h of absorption and washed twice with PBS and supplemented with medium. SARS-related-CoV WIV167 (link) and MERS-CoV HIV-pseudovirus were used as positive control for human/swine ACE2 or human/swine DPP4, respectively. After 24 h of infection, cells were washed with PBS and fixed with 4% formaldehyde in PBS (pH 7.4) for 20 min at room temperature. SARS-related-CoV WIV16 replication was detected using rabbit antibody against the SARS-related-CoV Rp3 N protein (made in house, 1:100) followed by Cy3-conjugated goat anti-rabbit IgG (1:50, Proteintech)7 (link). SADS-CoV replication was monitored using rabbit antibody against the SADSr-CoV 3755 N protein (made in house, 1:50) followed by FITC-conjugated goat anti-rabbit IgG (1:50, Proteintech). Nuclei were stained with DAPI (Beyotime). Staining patterns were examined using confocal microscopy on a FV1200 microscope (Olympus). Infection of MERS-CoV HIV-pseudovirus was monitored by luciferase 48 h after infection.
9
Immunofluorescence Analysis of Cell Cycle Proteins
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A. sinica at different developmental stages (0 h, 15 h and 3 d) were prepared and embedded in paraffin and sliced into 8 μm sections.
The sections were dewaxed and rehydrated before being washed in 0.2% TritionX-100 with PBS for 10 min. Paraffin sections were blocked with 3% BSA at room temperature for 2 h. The rabbit anti-As-CDC23 antibody (1:20 in PBST) was added and incubated overnight at 4 °C. Next day the sections were incubated in Cy3-conjugated goat anti-rabbit IgG (diluted 1:50; Proteintech) at 37 °C in the dark for 1 h, and washed with PBST again. Finally, the samples were stored in mounting medium with DAPI (4′, 6-diamidino-2-phenylindole; ZSGB-BIO, Beijing, China) and examined under confocal laser microscopy. The rabbit anti-As-CDC20 antibody and mouse anti-CDH1 antibody (Santa) were diluted 1:25 with PBST for double immunofluorescence antibodies, using FITC-conjugated goat anti-rabbit IgG (Proteintech) for CDC20 and TRITC-conjugated goat anti-mouse IgG (Proteintech) for CDH1 as the secondary antibodies.
The sections were dewaxed and rehydrated before being washed in 0.2% TritionX-100 with PBS for 10 min. Paraffin sections were blocked with 3% BSA at room temperature for 2 h. The rabbit anti-As-CDC23 antibody (1:20 in PBST) was added and incubated overnight at 4 °C. Next day the sections were incubated in Cy3-conjugated goat anti-rabbit IgG (diluted 1:50; Proteintech) at 37 °C in the dark for 1 h, and washed with PBST again. Finally, the samples were stored in mounting medium with DAPI (4′, 6-diamidino-2-phenylindole; ZSGB-BIO, Beijing, China) and examined under confocal laser microscopy. The rabbit anti-As-CDC20 antibody and mouse anti-CDH1 antibody (Santa) were diluted 1:25 with PBST for double immunofluorescence antibodies, using FITC-conjugated goat anti-rabbit IgG (Proteintech) for CDC20 and TRITC-conjugated goat anti-mouse IgG (Proteintech) for CDH1 as the secondary antibodies.
10
Intracellular Cytokine Profiling of Activated Splenocytes
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Single cell suspensions of spleen were lysed of Red Blood Cells, subsequently splenocyte were stimulated with plate-bound anti-CD3 5 μg/ml (eBioscience, Inc) and anti-CD28 2 μg/ml (eBioscience, Inc) in the presence of Leukocyte Activation Cocktail, with BD GolgiPlug™ (BD. Inc) for 6 hours. The collected cells were resuspended in 100 μl of 2% BSA in PBS incubated on ice for 10 min, fixed in 1 ml 2% paraformaldehyde at 4 °C for 1 hour. The cells were resuspended in 100 μl of 0.1% saponin (Sigma, Inc) to which 1:200 each of purified anti-IL-2 rabbit antibody (santa cruz, Inc) were added followed by incubation with 0.5 μg of FITC-conjugated goat anti-rabbit IgG (Proteintech, Inc). The Splenocytes were stained with PerCP/Cy5.5 anti-mouse IFN-γ (Biolegend, Inc) then incubated on ice water bath at 4 °C for 45 min, and washed with PBS containing 2% BSA. The cells were collected and analyzed using Fortessa or Accuri C6 and software (BD, Inc).
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