Immunoblotting was carried out as previously described (15 (link)) with diluted (1:1000) anti-PHGDH antibodies (HPA021241; Sigma), anti-HIF2α antibodies (ab51608; Abcam, Cambridge, MA, USA), anti-glyceraldehyde 3-phosphate dehydrogenase antibodies (GAPDH; MAB374, EMD Millipore, Billerica, MA), and anti-β-actin antibodies (bs-0061R; Bioss, Woburn, MA, USA). Immunohistochemistry were performed using an UltraVision Detection System (Thermo Scientific, Fremont, CA, USA) according to the manufacturer’s instructions. The primary rabbit monoclonal antibodies against Ki67 (ab92742; Abcam) were diluted 1:100. For immunofluorescence analyses, nuclei were stained with DAPI (1 μg/mL; Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA), and slides were mounted in Fluoromount (Diagnostic Biosystems, Pleasanton, CA, USA). Anti-PHGDH antibodies (HPA021241; Sigma) were used as the primary antibody at a dilution of 1:100, and binding was visualised using secondary antibodies conjugated to Alexa Fluor 488 (ab150077; Abcam).
4 6 diamidino 2 phenylindole (dapi)
Manufactured by LGC
Sourced in United States
DAPI is a fluorescent stain that binds to DNA. It is used in various laboratory applications to visualize and identify cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Merck Group (1 mentions)
Mab374, by Merck Group (1 mentions)
Bs 0061r, by Bioss Antibodies (1 mentions)
Ab92742, by Abcam (1 mentions)
Ab51608, by Abcam (1 mentions)
Hpa021241, by Merck Group (1 mentions)
Ultravision detection system, by Thermo Fisher Scientific (1 mentions)
Fluoromount, by Diagnostic BioSystems (1 mentions)
Ab150077, by Abcam (1 mentions)
Transwell cell culture insert, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Alexa fluor 488 or 555 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab6672, by Abcam (1 mentions)
Ab5076, by Abcam (1 mentions)
Anti γ tubulin gtu 88, by Merck Group (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Jb 4 resin, by Polysciences (1 mentions)
Fluorescent mounting media, by LGC (1 mentions)
Anti acetylated α tubulin 6 11b 1, by Merck Group (1 mentions)
6 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Immunoblotting and Immunohistochemistry Protocols
2
Transwell Migration Assay for Cell Motility
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The migration assay was performed as described previously [25 (link)], using transwell cell culture inserts (BD Bioscience, Franklin Lakes, NJ, USA) that were 6.5 mm in diameter with 8 μm pore filters. The cells (5.0 × 104 cells/well) were suspended in 350 μL of serum-free DMEM containing 0.1% bovine serum albumin (Sigma-Aldrich) and seeded into the upper well; 600 μL of 10% FBS-supplemented DMEM with or without 10 ng/mL MCP-1 and 100 ng/mL anti-MCP-1 neutralizing antibody (Abcam) was placed in the lower well of the transwell plate. After incubation for 6 h at 37°C under hypoxic conditions, cells that had not migrated from the upper side of the filter were scraped off with a cotton swab, and the membrane was fixed in 4% paraformaldehyde in PBS. After washing with PBS, the cells that had migrated onto the underside of the membrane were labeled with DAPI (1 : 1,000; Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) and counted under a fluorescence microscope in five high-power fields (400x magnification; Olympus IX70; Olympus Corp., Tokyo, Japan).
3
Immunofluorescence Imaging of Cellular Markers
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The cells that were plated onto poly‐lysine‐coating culture chamber slides (Matsunami, Osaka, Japan) were fixed with ice‐cold methanol for 10 minutes, washed in phosphate‐buffered saline (PBS) and incubated for 30 minutes with 1% of bovine serum albumin (BSA) in PBS with Tween (PBST) for blocking. The cells were then incubated overnight with primary antibodies directed against Iba1 (ab5076, Abcam), BRAF‐V600E (NBP2‐42702, Novus Bioscience), CSF‐1R (Abcam) and IL‐6 (ab6672, Abcam) and diluted in PBST containing 0.3% of BSA. The cells were then washed twice with PBS and incubated with Alexa Fluor 488‐ or 555‐conjugated secondary antibodies (Molecular Probes) for 1 h at room temperature in the dark. Nuclear staining was performed using DAPI (Kirkegaard & Perry Laboratories, Inc.). Images were captured using a confocal laser microscope (LSM780; Carl Zeiss AG).
4
Immunohistochemistry of Cellular Structures
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A previously published protocol was followed (44 (link),56 (link)). Primary antibodies were: Custom-made anti-Wtip (1:100 dilution; Covance, Inc., Denver, PA, USA), anti-γ-tubulin (GTU-88; 1:800 dilution; Sigma-Aldrich), anti-acetylated α-tubulin (6-11B-1; 1:800 dilution; Sigma-Aldrich), and anti-centrin (20H5; 1:1,000 dilution; EMD Millipore, Billerica, MA, USA). The following secondary antibodies were obtained from Thermo Fisher Scientific, Inc.: Goat anti-rabbit Alexa Fluor®546 (IgG [H+L]), goat anti-mouse Alexa Fluor®488 (IgG2b), goat anti-mouse Alexa Fluor®488 [IgG1 (γ1)], goat anti-mouse Alexa Fluor®488 [IgG2a (γ2a)] and goat anti-mouse Alexa Fluor®546 [IgG1 (γ1)]. Whole-mount immunohistochemistry samples were dehydrated with a graded series of methanol, embedded in JB4 resin (Polysciences, Inc., Warrington, PA, USA), and were cut into 5–7 µm sections using an RN2255 microtome (Leica Technology, Exton, PA, USA). The sections were stained with DAPI (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA), mounted in Fluorescent Mounting Media (Kirkegaard & Perry Laboratories, Inc.), and were imaged with a FV-1000 confocal laser-scanning microscope (Olympus America, Inc., Center Valley, PA, USA).
5
Quantitative Immunohistochemistry of Tumor Immune Markers
6
Immunofluorescence Staining in Cell Cultures
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After each of the following reactions, 2 wash steps for 10 minutes each in PBS
were performed. Cells at passage 4 were fixed with 2% paraformaldehyde in PBS
for 15 minutes. Cells were permeabilized with the help of 0.25% Triton-X100
(X100-5ML; Sigma-Aldrich) in PBS for 10 minutes. Blocking was achieved with 1%
BSA in PBS for 15 minutes. Primary antibodies were diluted according to
recommendations of the supplier in 1% BSA in PBS for 1 hour at 37 °C. Secondary
antibodies coupled with fluorochromes were applied at 1:500 dilution together
with DAPI (71-03-01; Seracare) at 1:1000 dilution in 1% BSA in PBS for 30
minutes at 37 °C. Observation was performed via fluorescence microscope
(BZ-X700; Keyence).
were performed. Cells at passage 4 were fixed with 2% paraformaldehyde in PBS
for 15 minutes. Cells were permeabilized with the help of 0.25% Triton-X100
(X100-5ML; Sigma-Aldrich) in PBS for 10 minutes. Blocking was achieved with 1%
BSA in PBS for 15 minutes. Primary antibodies were diluted according to
recommendations of the supplier in 1% BSA in PBS for 1 hour at 37 °C. Secondary
antibodies coupled with fluorochromes were applied at 1:500 dilution together
with DAPI (71-03-01; Seracare) at 1:1000 dilution in 1% BSA in PBS for 30
minutes at 37 °C. Observation was performed via fluorescence microscope
(BZ-X700; Keyence).
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