GYY4137 [morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate], DTT (DL-dithiothreitol), AMPK inhibitors Compound C (6-[4-[2-(1-piperidinyl)eth-oxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine), Ara-A (ATP-mimetic, 9-β-D-arabinofuranoside), and MMTS (S-methyl methane thiosulfonate) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Biotin-HPDP and HRP (horseradish peroxidase)-conjugated streptavidin were purchased from Thermo Scientific (Rockford, IL, United States). Primary antibodies for phospho-AMPKα (Thr172), AMPKα, phospho-CaMKKβ (Ser458/495), and CaMKKβ were bought from Cell Signaling Technology (Boston, MA, United States). Primary antibodies for NeuN (neuron-specific nuclear protein), c-Fos, and NPY were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Primary antibody for GAPDH (glyceraldehyde-3-phosphate dehydrogenase), HRP-conjugated secondary antibodies, fluorescent secondary antibodies, and bovine serum albumin (BSA) were obtained from Beyotime Biotechnology (Shanghai, China). Other agents were all purchased from commercial suppliers. Biotin-HPDP was freshly dissolved in dimethysulfoxide (DMSO) and other drugs were prepared freshly with double-distilled water or buffer solutions to the final concentrations before application.
Fluorescent secondary antibody
Manufactured by Beyotime
Sourced in China
Fluorescent secondary antibodies are laboratory reagents used in various immunoassay techniques. They are designed to bind to primary antibodies and amplify the signal, allowing for the detection and visualization of target molecules in samples.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (6 mentions)
Lsm 780, by Zeiss (2 mentions)
C fos, by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Gyy4137, by Merck Group (1 mentions)
Ara a, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Camkkβ, by Cell Signaling Technology (1 mentions)
Biotin hpdp, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Beyotime (1 mentions)
Anti aggrecan, by Merck Group (1 mentions)
Anti collagen 2, by GeneTex (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Media Cybernetics (1 mentions)
Leica dm4000b, by Leica Microsystems (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Lsm800 fluorescence microscope, by Zeiss (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
C myc, by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
13 protocols using fluorescent secondary antibody
1
AMPK Regulation Pathway Analysis
2
Chondrogenic Differentiation Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At day 14, cultured cells were washed three times with phosphate-buffered saline (PBS) and fixed for 10 min with 4% paraformaldehyde. Specimens were blocked with 5% bovine serum albumin for 1 h and incubated at 4°C overnight with the following primary antibodies: Anti-collagen II (1:100; GeneTex, Irvine, CA, USA), anti-aggrecan (1:200; Millipore, Billerica, MA, USA) and anti-SRY (sex determining region Y)-box 9 (SOX9) (1:200; Abcam, Cambridge, UK). Subsequent to washing three times with PBS, the cells were incubated with fluorescent secondary antibodies (Beyotime Institute of Biotechnology, Shanghai, China) for 2 h. For nuclear staining, DAPI (Beyotime Institute of Biotechnology) was applied for 3 min and subsequently observed under a fluorescence microscope (Leica DM 4000 B; Leica Microsystems, Wetzlar, Germany). Cell counting of 1,000 cells in five randomly selected fields was performed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc. Rockville, MD, USA) and the percentage of positively stained cells was calculated as the ratio of the number of positive cells to the total number of DAPI-positive cells.
3
Protein Expression and Localization Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, proteins were prepared by radioimmunoprecipitation assay (RIPA) buffer and quantified by a BCA Protein Assay Kit (Beyotime, China). The nuclear protein was obtained using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) following the manufacturer’s protocol. Samples were subjected to 10% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked and then sequentially incubated with the primary antibody, the secondary antibody, and detected by chemiluminescence. The antibodies used included primary antibodies against RUNX3, E-cadherin, Vimentin, N-cadherin, β-catenin, c-Myc, CD44, CCND1, GAPDH (glyceraldehyde-3-phosphate dehydrogenase, as negative control), and β-actin (Proteintech, USA). IF assays were conducted as reported previously.12 (link) In brief, cells grown on slides, including the transfected GC cells, were successively incubated with primary antibodies (against E-cadherin and Vimentin) and fluorescent secondary antibodies (Beyotime) and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (nucleus). Cells on the slides were observed and imaged using a ZEISS LSM800 fluorescence microscope (Carl Zeiss AG, Germany).
4
Visualization of ZDHHC2 and AGK in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were incubated with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt (DiD, Far-red Plasma Membrane Fluorescent Probe, Beyotime, C1039) for 20 minutes to label the cell membrane. Then, the cells were fixed with paraformaldehyde for 15 minutes and permeabilized with 0.2% Triton X-100 for 10 minutes. Then, the cells were incubated with anti-ZDHHC2 and anti-AGK antibodies at 4°C overnight. The next day, the cells were incubated with fluorescent secondary antibodies (Beyotime) for 1 hour, followed by washing with PBS buffer for 3 times. Then DAPI (Beyotime) was used to stain cell nuclei for 10 minutes. After washing with PBS buffer for 3 times, the samples were analyzed through confocal microscopy (Andor, Dragonfly, 63× objective lens).
5
Immunofluorescence Staining of Brain Tissue and Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The brain tissue and cell sections were prepared. After permeabilization with 0.3% Triton X-100 (Sigma-Aldrich), sections were probed with LAMP-2A (1:100, Abcam, ab18528), N-CoR (1:100, Novus, NBP1-28863) for tissues or N-CoR (1:100, Santa Cruz, sc-1609) for cells. After being washed with PBS containing 0.05% of Tween-20 (Sigma-Aldrich), sections were incubated with fluorescent secondary antibodies (Beyotime, China) and antifade mounting medium containing 1:500 DAPI (Beyotime, China). Leica fluorescent microscope was used for visualization.
6
Integrin and Inflammasome Immunostaining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue sections were blocked with 3% BSA for 30 min at 25 °C. Sections were incubated overnight at 4 °C with antibodies against integrin αVβ5 (1:100, Santa Cruz Biotechnology, Japan). For GC and TC staining, sections were fixed in 4% paraformaldehyde (Servicebio, China) for 30 min at 25 °C and then permeabilized with 0.3% Triton X-100 (Beyotime, China). After washing with PBS three times, the cells were blocked with 3% BSA for 30 min at 25 °C. Cells were incubated with antibodies against IRE1α (1:100, Proteintech), apoptosis-associated speck-like protein containing a CARD (ASC, 1:100, AdipoGen Life Science, USA), NLRP3 (1:100, CST, USA), a-smooth muscle actin (α-SMA, 1:100, Abcam, UK) and collagen I (1:100, Bioworld Technology, China) overnight at 4 °C. After washing with PBS three times, tissue sections and cells were incubated at 25 °C for 2 h with fluorescent secondary antibodies (Beyotime). Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime) at a dilution of 1:2000 for 30 min and photographed using an Olympus laser scanning confocal microscope (FV3000, Japan).
7
Plasma Membrane Protein Localization by Immunofluorescence
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were incubated with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindodicarbocyanine,4‐chlorobenzenesulfonate Salt (DiD, Far‐red Plasma Membrane Fluorescent Probe, Beyotime, C1039, China) for 20 min to label the cell membrane. Then, the cells were fixed with paraformaldehyde for 15 min and permeabilized with 0.2% Triton X‐100 for 10 min. Next, the cells were incubated with primary antibodies at 4 °C overnight. The following day, the cells were incubated with fluorescent secondary antibodies (Beyotime, China) for 1 h, and washed three times with PBS buffer. Then DAPI (Beyotime, China) was used to stain cell nuclei for 10 min. After washing with PBS buffer for three times, the samples were analyzed through confocal microscopy.
8
Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 24-well plate was positioned with a slide at the bottom, and 5 × 104 cells were planted to each well. After culture for 24 h, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% TritonX-100 and then blocked with immunol staining blocking buffer (Beyotime, Shanghai, China) for 30 min. The cells were next incubated with primary antibody overnight at 4℃. Next day, following 2 h of fluorescent secondary antibody (Beyotime, Shanghai, China) incubation, the nuclei were stained with DAPI (Beyotime, Shanghai, China) for 15 min. The pictures were captured under confocal laser scanning microscopy (Zeiss, Jena, Germany).
9
Muscle Glycogen and AMPK Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The muscle sample was fixed in a 4% paraformaldehyde solution, and the muscle tissue of appropriate size was embedded in paraffin. It is cut into 3–5 μm laminas and dyed with periodic acid Schiff (PAS) staining method from Servicebio in Wuhan, China. Nikon TS100 optical microscope was used for observation and Image J software was used to confirm muscle glycogen.
According to the SABC kit, the embedded paraffin samples were sliced and put into the oven at 60 °C for 1–2 h, after rehydration, inactivation, and antigen repair for washing, the slices were closed with goat serum sealing solution (5% BSA) for 1 h at indoor temperature and then hatched with P-AMPK (1:100, HUABIO, SD0810) primary antibody at 4 °C overnight. Dropwise addition of fluorescent secondary antibody (1:1000, Beyotime, China) was incubated at room temperature and protected from light for 1 h, stained with DAPI, blocked, and photographed for analysis with an inverted fluorescence microscope. The cumulative optical density (IOD) was quantified using Image Pro Plus® 4.5.
According to the SABC kit, the embedded paraffin samples were sliced and put into the oven at 60 °C for 1–2 h, after rehydration, inactivation, and antigen repair for washing, the slices were closed with goat serum sealing solution (5% BSA) for 1 h at indoor temperature and then hatched with P-AMPK (1:100, HUABIO, SD0810) primary antibody at 4 °C overnight. Dropwise addition of fluorescent secondary antibody (1:1000, Beyotime, China) was incubated at room temperature and protected from light for 1 h, stained with DAPI, blocked, and photographed for analysis with an inverted fluorescence microscope. The cumulative optical density (IOD) was quantified using Image Pro Plus® 4.5.
10
Caspase-11 Immunofluorescence Staining in MH-S Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MH-S cell were placed on slides, fixed with 4% paraformaldehyde at room temperature for 30 min, then washed with PBS for 2–3 times. Placed the cell slides in 1% Triton x-100 (JISSKANG, China) for 10 min. After washing with PBS, the cell slides were blocked in 5% BSA for 1 h. Then the diluted anti-mouse caspase-11(ab240991, abcam, 1:100) was added and incubated overnight at 4 °C. On the second day, incubate the cell slides with the fluorescent secondary antibody (Beyotime, China) at 37 °C for 1 h under the dark environment. After rinsing, fluormount with DAPI (AntGene, China) was added to stain the nucleus. Finally, seal the tablet and observe the slides under the fluorescence microscope.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!