The xenograft tumors were fixed and cut into 5-μm sections, which were dewaxed, rehydrated, and fixed on glass slides. Each slide was dropped with 3% H2O2 for 15 min and then blocked with normal goat-serum (Solarbio) at 23–25°C for 15 min. After that, the sections were reacted with the antibodies against NF-κB p65 (1:100, sc-8008, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and SNAIL (1:500, ab224731, Abcam) overnight at 4°C, and then with IgG (1:2,000, ab205719, Abcam) for 15 min at 37°C. The sections were further reacted with 40 μL horseradish peroxidase-labeled streptavidin-working solution at 23–25°C for 15 min. Thereafter, the sections were counter-stained by hematoxylin, dehydrated, sealed, and observed under the microscope.
Ab224731
Manufactured by Abcam
Sourced in United States
Ab224731 is a recombinant anti-SARS-CoV-2 spike protein antibody. It is produced in HEK293 cells.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Ab205719, by Abcam (1 mentions)
Sc 8008, by Santa Cruz Biotechnology (1 mentions)
Normal goat serum (ngs), by Solarbio (1 mentions)
Srsf1, by Abcam (1 mentions)
Vimentin, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ab15007, by Abcam (1 mentions)
Ab179483, by Abcam (1 mentions)
Goat anti mouse igg h l alexa fluor 488, by Abcam (1 mentions)
Ab192890, by Abcam (1 mentions)
Ab150113, by Abcam (1 mentions)
Anti rabbit igg h l alexa fluor 647, by Abcam (1 mentions)
Ab195352, by Abcam (1 mentions)
Ab137321, by Abcam (1 mentions)
Ab207301, by Abcam (1 mentions)
Ab18203, by Abcam (1 mentions)
Ab40772, by Abcam (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Roche (1 mentions)
6 protocols using ab224731
1
Immunohistochemical Analysis of NF-κB p65 and SNAIL
2
Immunohistochemical Analysis of Epithelial-Mesenchymal Transition
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Tissue blocks fixed in 4% paraformaldehyde were extracted, embedded in paraffin, and sectioned into 4-μm-thick sections. Then the paraffin-embedded sections were subjected to IHC staining using standard procedures. Sections were incubated with primary antibody followed by incubation with HRP-labeled secondary antibody. The diluted concentrations of antibodies used in IHC were as follows: Snail and Slug (1: 5000, ab224731), Vimentin (1: 200, ab92547) antibodies were from Abcam; SRSF1 (1: 500, PA5-30,220) antibody was from Thermo Fisher. The binding extent of the antibodies was visualized using DAB staining. Tissue sections were re-stained with hematoxylin. The cross-sectional images were taken by LeicaMicrosystems (model: DM2000, CMSGmbH, Wetzlar, Germany). Six fields per section were randomly selected for immunohistochemical scoring by ImagePro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
3
Immunofluorescence Analysis of Autophagy Markers
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The LC3B expression that contributes to autophagy was detected by immunofluorescence (IF). Briefly, 1 × 106 HPAEpiCs were fixed with 4% paraformaldehyde for 30 min at room temperature in 24-well plates. Next, the cells were blocked with 10% goat serum for 15 min at 4°C and, subsequently, incubated with an LC3B antibody (1 : 200, ab192890, Abcam), SLUG antibody (1 : 300, ab224731, Abcam), and HIF-1α antibody (1 : 500, ab179483, Abcam). The cells were then incubated with the TRITC-conjugated secondary antibody Anti-Rabbit IgG H&L, (Alexa Fluor ® 647) (1 : 1000, ab15007, Abcam) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 488, 1 : 1000, ab150113, Abcam) for 1 h at 37°C in the dark. After being counterstained with 4′,6-diamidino-2- phenylindole (DAPI, 0.1 μg/mL; Sigma Aldrich, St. Louis, MO, USA) for 5 min, the cells were photographed using a fluorescent microscope at 647 nm (OLYMPUS, IX71).
4
Immunohistochemical Analysis of EMT Markers
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The tumor tissue samples were fixed with 10 % neutral formalin solution, dehydrated, embedded in paraffin, and sectioned by an ultrathin sectioning machine. The sections were then dewaxed with xylene, rehydrated with graded alcohol, and incubated with 3 % hydrogen peroxide to block the activity of endogenous peroxidase. The sections were boiled in 10 mM sodium citrate (pH 6.0) for 30 min, sealed in 10 % normal goat serum for 15 min, and then incubated overnight with antibodies against METTL3 (1:500, ab195352, Abcam), HMGA2 (1:500, ab207301, Abcam), Snail (1:2000, ab224731, Abcam), N-cadherin (1:1000, ab18203, Abcam), and Vimentin (1:500, ab137321, Abcam) in a wet chamber at 4 ℃. The next day, the sections were washed with PBS and incubated with secondary antibody for 1 h at room temperature prior to visualization as above.
5
Immunohistochemical Analysis of Epithelial-Mesenchymal Transition Markers
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Tumor tissues were fixed in 4% paraformaldehyde for 24 h at 4˚C, embedded in paraffin and then cut into 4-µm sections. Next, the sections were heated at 60˚C, dewaxed using the xylene reagent and rehydrated (100, 100, 95, 90, 80 and 70% alcohol for 5 min, respectively). Next, 3% H2O2 solution was dropped onto the slices for 10 min. Subsequently, the tissues were blocked in 5% BSA (Roche Diagnostics) for 20 min at room temperature, and then incubated with primary antibodies, including anti-N-Cadherin (1:100), anti-Snail (1:100; cat. no. ab224731; Abcam) and anti-E-Cadherin (1:100; cat. no. ab40772; Abcam) at 4˚C overnight. Next, the tissues were incubated with secondary antibody (HRP-conjugated anti-rabbit IgG; 1:500;) at 37˚C for 50 min. Immunostaining was detected by adding 3,3'-diaminobenzidine for 30 sec. Images were observed using a light microscope.
6
Nuclear Morphology and Protein Levels in SK-N-SH Cells
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The nuclear morphologies of the SK-N-SH cells were visualized by immunostaining with lamin A and lamin B1. The lamin A and vimentin levels in the cells were characterized by immunostaining of lamin A and vimentin. Cells were cultured on nanopillars and incubated overnight before fixation with prewarmed 4% paraformaldehyde (PFA) in PBS (Boster biological technology AR1068) for 15 minutes at room temperature. After washing with PBS three times, the cells were permeabilized in 0.5% Triton X-100 (Sigma) in PBS for 15 minutes, followed by blocking with 5% bovine serum albumin (BSA) (Sigma) in PBS for 1 hour. Subsequently, the samples were incubated with primary antibodies (anti-lamin A, Abcam, ab26300; anti-lamin B1, Abcam, ab16048; anti-vimentin, Sigma, V5255; anti-SNAIL + SLUG, Abcam, ab224731) at 1 : 400 dilution at room temperature for 1 hour or 4 °C overnight. The samples were again washed with PBS for three times and then incubated with secondary antibodies (chicken anti-rabbit IgG Alexa 488, Invitrogen, A21441; anti-mouse IgG Alexa 555, Cell Signaling Technology, 4409s) at 1 : 600 dilution at room temperature for 1 hour. After washing with PBS three times, the samples were stained with DAPI (Sigma), phalloidin (Cytoskeleton, Inc.) or cellmask (Invitrogen) in different experiments.
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