Cells were seeded evenly on the confocal dish at a density of 5 × 104 per well for 48 h and then probed with primary antibodies against TUSC3 (#SAB4503183, Proteintech, China). Next, the coverslips were incubated with fluorescein isothiocyanate (FITC)-conjugated goat antibodies against rabbit IgG (anti-rabbit IgG, #ab6940, Abcam, MA). From then on, the dishes were protected from light. After washing with PBS in a dark place, the dishes were incubated with primary antibody against LIPC (#3538, Proteintech, China), and then incubated with rhodamine-conjugated goat antibodies against rabbit IgG (anti-rabbit IgG, #ab6940, Abcam, MA). Following counterstaining with 4′,6-diamidino-2-phenylindole (DAPI, Sigma, MO), images were captured using an Olympus FV1000 confocal laser-scanning microscope (Olympus America Inc., NY).
Ab6940
Manufactured by Abcam
Sourced in United Kingdom
Ab6940 is an anti-GAPDH rabbit monoclonal antibody from Abcam. It is designed for use in Western blotting and immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ap124c, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Ab1416, by Abcam (1 mentions)
Ab55006, by Abcam (1 mentions)
Ab45939, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Nitrocellulose nc membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Boster Bio (1 mentions)
Ab179467, by Abcam (1 mentions)
Ab185736, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Goat anti rabbit igg h l secondary antibody, by Abcam (1 mentions)
Anti hmga2, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
11 protocols using ab6940
1
Immunofluorescence Staining of TUSC3 and LIPC
2
FOXM1 Knockdown in OSCC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OSCC cells were plated in six-well plates and FOXM1 was knocked down. The cells were collected, suspended in lysis buffer (50 mmol/l Tris-HCl, pH 6.8, 100 mmol/l DTT, 2% SDS, 0.1% bromophenol blue and 10% glycerol) on ice for 5–10 min and centrifuged at 4°C (12,000 × g, 15 min). The protein concentrations were determined using a bicinchoninic acid protein assay. The 50 µg protein samples per lane were separated by 10% SDS-PAGE and transferred onto nitrocellulose filter membranes. The membranes were blocked in freshly prepared PBS containing 5% non-fat dried milk for 2 h at room temperature. The blots were subsequently probed with primary antibodies specific for FOXM1, E-cadherin, vimentin and β-actin (1:1,000; ab55006, ab1416, ab45939 and ab8226 respectively; Abcam,) overnight at 4°C. The membranes were washed three times with PBS-0.05% Tween 20 and incubated with the corresponding secondary antibodies (1:1,000; ab6940; Abcam) for 1 h at room temperature. The blots were subsequently incubated in the dark for enhanced chemiluminescence and visualized through exposure to ECL reagents (GE Healthcare, Chicago, IL, USA).
3
Western Blot Analysis of TCF4 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1 × 106 cells were washed with PBS, and lysed using 200 µl RIPA lysis buffer (Beyotime, Hangzhou, China). The supernatant was collected after high speed centrifugation (16,000×g, 10 min, 4 °C) and the protein was denatured by heating the samples in boiling water for 5 min. After quantifying the protein by the bicinchoninic acid (BCA) Protein Assay Kit (Boster, Wuhan, China), protein samples were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred into a nitrocellulose (NC) membrane (Millipore, MA, USA). Then, the NC membrane was blocked with defatted milk for 30 min at room temperature. Then the primary antibody was added on the NC membrane and incubated overnight at 4 °C. After that, the NC membrane was washed with TBST, followed by being incubated with the secondary antibody for 1 h at room temperature, and then developed chemiluminescence with ECL reagent (Millipore, Bedford, MA, USA). The antibodies used in this study were: anti-TCF4 antibody (ab185736, 1: 500, Abcam, Cambridge, UK), anti-β-actin antibody (ab179467, 1:2000, Abcam, Cambridge, UK), and Goat polyclonal Secondary Antibody to Rabbit IgG–H&L (ab6940, 1:2000, Abcam, Cambridge, UK).
4
Western Blot Analysis of UCP2 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blots were performed with protein samples as indicated. After transfer to a nitrocellulose membrane samples were incubated with either an antibody directed against UCP2 and subsequently with an antibody directed against actin or voltage-dependent anion exchanger (VDAC) or GAPDH. Signals were detected by a fluorescence coupled secondary antibody (Abcam, ab6940). Western blots dealing with UCP2 expression were performed with an antibody evaluated before.18 (link) Specificity was tested in tissue samples from spleens of UCP2−/− mice and wild-type mice, respectively (Supplementary material online , Figure S1 ). Control peptide for UCP2 was a recombinant human UCP2.19 (link)
5
Immunofluorescent Labeling of Adipose Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We followed a previously reported protocol 100 (link). Fresh adipose tissues were fixed with 4% paraformaldehyde (PFA) overnight at room temperature. Fixed tissues were cut into 2-3 mm3 pieces, blocked with 5% BSA for 1 hour at room temperature, and incubated with FITC anti-mouse F4/80 Antibody (123107, Biolegend, 1:100) or Alexa Fluor 647 anti-mouse F4/80 antibody (123122, Biolegend, 1:100) or Anti-prosaposin (PSAP) antibody (ab180751, Abcam, 1:100) at 4ºC overnight, followed by Cy2 conjugated secondary antibody (ab6940, Abcam, 1:500) at room temperature for 1 hour. Observation of treated fat tissues was performed on TPFM. F4/80 is a major marker of macrophages 101 (link), and prosaposin (PSAP) is a marker of lipofuscin 102 (link), 103 (link). Both F4/80 and PSAP are used to label macrophages.
6
Western Blot Analysis of HMGA2 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with PBS, and lysed using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) for 10 min on ice. The BCA method was used for protein quantification. Following centrifugation at 14,000 × g for 15 min at 4°C, the supernatant was collected. Proteins (30 µg) were separated by SDS-PAGE (12% gel), and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Membranes were washed with Tris-buffered saline containing Tween-20 (TBST), and blocked with 5% skimmed milk with TBST at 25°C for 1 h. Membranes were then incubated with monoclonal primary antibody at 4°C overnight. The following primary antibodies were used: Anti-HMGA2 (1:500; catalog no. ab52039; Abcam, Cambridge, UK) and anti-GAPDH (1:500; catalog no. ab9485; Abcam). Membranes were washed with TBST for 30 min and incubated at 25°C for 1 h with goat anti-rabbit IgG H&L secondary antibody (1:1,000; catalog no. ab6940; Abcam). Enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc.) was used to detect the signal on the membrane.
7
Western Blot Analysis of Hepatocellular Carcinoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins from HCC cells were extracted using radioimmunoprecipitation assay
lysis buffer and qualified by a bicinchoninic acid assay detecting kit (Thermo
Fisher Scientific, Waltham, MA, USA). Proteins (20 μg) were boiled at 100°C in
sodium dodecyl sulfate (SDS) sample buffer for 5 min and separated with
SDS-polyacrylamide gel electrophoresis and then transferred onto a
polyvinylidene difluoride membrane. After blocking, membranes were incubated
with primary anti-ROCK1 (ab134181, Abcam, Cambridge, UK; 1:1,000), anti-cylinD1
(ab251892, Abcam, 1:500), anti-p21 (ab218311, Abcam, 1:1,000), anti-E-cadherin
(ab194982, Abcam, 1:500), and anti-MMP2 (ab97779, 1:1,000) antibodies for
overnight. The following day, secondary anti-rabbit or anti-mouse antibodies
(ab6940, ab97035, Abcam, 1:2,000) were added and incubated with membranes for 1
h at room temperature. The captured bands were quantified using Image Lab™
Software (Bio-Rad, Hercules, California, USA).
lysis buffer and qualified by a bicinchoninic acid assay detecting kit (Thermo
Fisher Scientific, Waltham, MA, USA). Proteins (20 μg) were boiled at 100°C in
sodium dodecyl sulfate (SDS) sample buffer for 5 min and separated with
SDS-polyacrylamide gel electrophoresis and then transferred onto a
polyvinylidene difluoride membrane. After blocking, membranes were incubated
with primary anti-ROCK1 (ab134181, Abcam, Cambridge, UK; 1:1,000), anti-cylinD1
(ab251892, Abcam, 1:500), anti-p21 (ab218311, Abcam, 1:1,000), anti-E-cadherin
(ab194982, Abcam, 1:500), and anti-MMP2 (ab97779, 1:1,000) antibodies for
overnight. The following day, secondary anti-rabbit or anti-mouse antibodies
(ab6940, ab97035, Abcam, 1:2,000) were added and incubated with membranes for 1
h at room temperature. The captured bands were quantified using Image Lab™
Software (Bio-Rad, Hercules, California, USA).
8
Chlamydia muridarum Visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primary antibody: A rabbit antibody (made with purified C. muridarum elementary bodies) was gift from Jingyue Ma in Tianjin Medical University General Hospital. After primary antibody incubation, the secondary antibody (goat anti-rabbit IgG conjugated with Cy2, #ab6940, Abcam) was added and the nuclei was visualized by DNA dye Hoechst 33258 (#ab228550, Abcam). The doubly labeled samples were used for counting for chlamydial inclusions under a fluorescence microscope (AX70, Olympus) with a CCD camera (Hamamatsu).
9
Quantifying ER-alpha and HIF-1alpha Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expressions of HIF-1alpha and ER-alpha were examined by Western Blot (n = 3). The resulting blots were probed with rabbit monoclonal (#EPR4097, Estrogen Receptor alpha, Abcam, UK), rabbit polyclonal (#ab82832, Anti-HIF-1 alpha, Abcam, UK), and secondary antibodies (#ab6940, Goat polyclonal Secondary Antibody to Rabbit IgG; H&L, Abcam, UK), respectively (Fig. 4, A-1 & A-2). The expressions of ER-alpha (1:200 dilution; ZETA, Bosterbio, USA) for MCF-7CH and MCF-7 were also examined by immuno uorescence assay for three times. Fluorescent gathered in the nucleus of ER positive cells. Photos were taken for more than 100 cells before analyzed by ImageJ (Fig. 4, B-1 & B-2).
10
Structured Illumination Microscopy for nSB Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Structured illumination microscopy was performed as described previously [15] . Briefly, a DIG-labeled HSATIII ASO was used to detect nSBs. The DIG-labeled probes were detected using an anti-DIG monoclonal antibody (Abcam, 21H8, ab420, 1:100 dilution) and a
Cy3-conjugated anti-mouse secondary antibody (Millipore, AP124C, 1:100 dilution).
SAFB-positive and HNRNPM-positive cells were visualized using anti-SAFB and anti-HNRNPM antibodies, respectively, and a Cy2-conjugated secondary antibody (Abcam, ab6940, 1:100 dilution). Images were captured using a ELYRA PS.1 microscope (Zeiss) with a 100× objective lens, as described previously [15] .
Cy3-conjugated anti-mouse secondary antibody (Millipore, AP124C, 1:100 dilution).
SAFB-positive and HNRNPM-positive cells were visualized using anti-SAFB and anti-HNRNPM antibodies, respectively, and a Cy2-conjugated secondary antibody (Abcam, ab6940, 1:100 dilution). Images were captured using a ELYRA PS.1 microscope (Zeiss) with a 100× objective lens, as described previously [15] .
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!