The materials used included hexamethyldisiloxane (HMDS, Sigma-Aldrich, St. Louis, MO, USA); sodium fluoride (NaF, Sigma Chemical, St. Louis, MO, USA); trypsin (Trypsin Gold Mass Spectrometry, Promega, Madison, USA); PlusOne 2D Cleanup kit (GE Healthcare, Uppsala, Sweden) and 3 kDa AMICON (Millipore, St Charles, MO, USA). Antibodies: nNOS (H-299, sc-8309 Santa Cruz, Dallas, TX, USA), Mouse anti-HuC/D (A-21271, Invitrogen, Waltham, MA, USA), Rabbit anti-CGRP (AB15360, Millipore, St Charles, MO, USA), Rabbit anti-VIP (V0390, Sigma-Aldrich, St. Louis, MO, USA), Goat anti-substance P (sc9758, Santa Cruz, Dallas, TX, USA), Anti-rabbit 546 (A10040, Invitrogen, Waltham, MA, USA), Anti-mouse 488 (A21202, Invitrogen, Waltham, MA, USA), Anti-rabbit 546 (A10040, Invitrogen, Waltham, MA, USA), and Anti-goat 568 (A11057, Invitrogen, Waltham, MAs, USA).
A10040
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A10040 is a laboratory equipment product offered by Thermo Fisher Scientific. It is a core function device designed for scientific and research applications. No further details are provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
A21202, by Thermo Fisher Scientific (14 mentions)
A10036, by Thermo Fisher Scientific (9 mentions)
A21206, by Thermo Fisher Scientific (8 mentions)
A11055, by Thermo Fisher Scientific (7 mentions)
Lsm 700 confocal microscope, by Zeiss (4 mentions)
A11029, by Thermo Fisher Scientific (4 mentions)
A31571, by Thermo Fisher Scientific (4 mentions)
A11058, by Thermo Fisher Scientific (3 mentions)
A31573, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
A11001, by Thermo Fisher Scientific (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Ab6640, by Abcam (2 mentions)
Prolong gold antifade reagent with dapi, by Cell Signaling Technology (2 mentions)
A21207, by Thermo Fisher Scientific (2 mentions)
A21203, by Thermo Fisher Scientific (2 mentions)
Ddx25 sc 51271, by Santa Cruz Biotechnology (2 mentions)
Gapdh 5g4, by HyTest (2 mentions)
A 11056, by Thermo Fisher Scientific (2 mentions)
A21447, by Thermo Fisher Scientific (2 mentions)
42 protocols using a10040
1
Immunofluorescence Analysis of Neuronal Markers
2
DHA-induced vWF Expression in HUVECs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HUVECs were grown on an adhesive culture dish (Sigma‐Aldrich) and then treated with 25 μM DHA. After 24 h of stimulation, the cells were washed and fixed for 10 min with 4 % paraformaldehyde and then blocked with 5 % bovine serum albumin. Next, the samples were incubated with a rabbit polyclonal antibody anti‐human vWF (1:200, A008229, Dako) for one night at 4 °C and incubated with an Alexa Fluor 546 secondary antibody (A10040, Life Technologies) for 2 h at normal temperature. The nuclei were stained with DAPI (Roche; Mannheim, Germany). The samples were pictured with an Olympus FSX100 Imaging System (Olympus Corporation, Tokyo, Japan), and the excitation wavelength was 546 nm.
3
Characterization of Cancer Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human oral squamous cancer cell line OSC–19 [34 ] was a generous gift from Dr Faye Johnson, MD Anderson Cancer Center, Houston, USA. Human glioblastoma cell line U–87 MG was purchased from American Type Culture Collection (ATCC; LGC Standards, Middlesex, UK). Prior to use, both cell lines were maintained as monolayers in RPMI 1640 growth media (R7388) supplemented with 10% (v/v) fetal bovine serum (FBS)(F1051), 1 mM sodium pyruvate (S8636) and 2 mM L-glutamine (G7513) (All from Sigma–Aldrich, Dorset, UK). For the remainder of the manuscript, RPMI 1640 growth media supplemented with FBS is referred to as “complete” whereas RPMI 1640 growth media not supplemented with FBS is referred to as “incomplete”. When required, cells were trypsinized and used as a suspension in growth media.
Fluorescein diacetate (FDA) (Sigma F7378), Rhodamine B (Sigma, R6626) propidium iodide (PI) (Sigma P4170), Cascade Blue®-10 kDa and Tetramethylrhodamine (TRITC)-70 kDa dextrans (Life Technologies, D1976 and D1818) were used as solutions in phosphate-buffered saline (PBS) (Lonza BE17-516F). PBS 5X was prepared by dissolving of 1 pack of PBS powder (Sigma P3813) in 200 ml deionised water. For immunofluorescence primary anti-Ki–67 antibody (abcam ab92742), secondary donkey anti-rabbit alexa-546-conjugated (life technologies A10040) and DAPI (life technologies D1306) were used.
Fluorescein diacetate (FDA) (Sigma F7378), Rhodamine B (Sigma, R6626) propidium iodide (PI) (Sigma P4170), Cascade Blue®-10 kDa and Tetramethylrhodamine (TRITC)-70 kDa dextrans (Life Technologies, D1976 and D1818) were used as solutions in phosphate-buffered saline (PBS) (Lonza BE17-516F). PBS 5X was prepared by dissolving of 1 pack of PBS powder (Sigma P3813) in 200 ml deionised water. For immunofluorescence primary anti-Ki–67 antibody (abcam ab92742), secondary donkey anti-rabbit alexa-546-conjugated (life technologies A10040) and DAPI (life technologies D1306) were used.
4
Immunofluorescence Analysis of HA and IFITM3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For IF analysis of HA and IFITM3 localization, A549 or 293T cells were grown on glass coverslips in 24-well plates. Cells were either transfected with expression plasmids for HA (A/WSN/33) and IFITM3 for 24 h or infected with A/WSN/33 (MOI 1) for 16 h. Cells were fixed with 3% paraformaldehyde for 15 min, washed, and permeabilized using IF buffer (PBS supplemented with 50 mM NH4Cl, 0.1% saponin, and 2% BSA). Cells were incubated for 1 h at room temperature with primary antibodies (rabbit polyclonal anti-IFITM3 and mouse monoclonal anti-A/WSN/33 HA clone H15-B9-22) and washed three times with IF buffer before secondary antibodies (A-21202 and A10040; Life Technologies) were added for 1 h at room temperature. Cells were washed three times with IF buffer, inversely mounted onto glass microscope slides using DAPI Fluoromount G (#0100-20; Southern Biotech), and images were acquired with a confocal laser scanning microscope (Leica SP5).
5
Antibody Validation for Western Blot and IHC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used were Mer for WB (AF591; R&D Systems), Mer for IHC (DS5MMER; eBioscience), Axl for IHC (AF854; R&D Systems), Axl for immunoprecipitation (M-20; Santa Cruz), GAPDH (MAB374, clone 6C5; Millipore), phospho-tyrosine (clone 4G10; Millipore), Gas6 (AF986; R&D Systems), CD31 (ab28364; Abcam), MARCO (MCA1849; AbD Serotec), Laminin (L9393; Sigma-Aldrich), Desmin (ab32362; Abcam), cleaved Caspase 3 (Asp175; Cell Signaling), F4/80 (MCA497; AbD Serotec), and Ki67 (65241; BioLegend). Axl, Mer, and Gas6 antibody specificity for immunohistochemistry, immunocytochemistry, immunoprecipitation, and immunoblotting was tested using samples from corresponding mouse mutants (e.g., Fig 1G and H ). Secondary antibodies used for immunoblot analysis were horseradish-peroxidase-conjugated anti-goat (705-035-003) from Jackson ImmunoResearch and antimouse (NA931V) and antirabbit (NA934V) from GE Healthcare. Secondary antibodies for immunocyto- and immunohistochemistry were fluorophore-conjugated antigoat (A-11055 from Life Technologies, or 705-166-147 from Jackson ImmunoResearch), antirabbit (A-10040 or A-21206 from Life Technologies), antirat (712-545-153 or 712-165-153 from Jackson ImmunoResearch), and antimouse (A-11029 from Life Technologies, or 715-166-150 from Jackson ImmunoResearch).
6
Immunohistochemistry for Hair Cells and Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole mount tissues or cryosections were blocked with 5% donkey serum, 0.1% Triton-X 100, 1% BSA, and 0.02% sodium azide (NaN3) in PBS at pH 7.4 for 1 hour at room temperature (RT). Samples were then incubated in primary antibodies overnight at 4 °C. The following primary antibodies were employed: rabbit anti-myosin 7a (1:200; 25-6790; Proteus BioSciences) for hair cells labeling, rat anti-F4/80 (1:150, ab6640, Abcam Inc., Cambridge, MA, USA) for macrophages labeling [18, 22] . The specimens were incubated with secondary antibodies diluted in 0.1% Triton-X 100, 0.1% BSA and 0.02% NaN3 in PBS for 1 hour at RT. The secondary antibodies were conjugated with Alexa Fluor 546 (1:500; A10040, Life Technologies, Carlsbad, CA). After washing with PBS, specimens were mounted in ProLong ® Gold Antifade Reagent with DAPI (Cell signaling, #8961 Danvers, MA 01923) and placed under a cover slip. Images were captured using a LSM700 confocal microscope (Zeiss, Germany) at 10X magnification.
7
Immunohistochemical analysis of neurogenesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BrdU was from Sigma-Aldrich. Antibodies used were as follows: anti-mouse Axl (R&D AF854), anti-Mer (eBioscience DS5MMER and R&D AF591), anti-mouse Gas6 (R&D AF986), anti-BrdU (BU1/75 [ICR1], AbD serotec), anti-Iba1 (Wako 019-19741, 1:200, and Novus NB100-1028), anti-cleaved Casp3 (Asp175, Cell Signaling 9661), anti-doublecortin (DCX, Millipore ab2253), anti-ZnT3 (Sysy 197 002), anti-PSD-95 (Abcam ab18258), anti-CD68 (BioRad MCA1957), anti-NeuN (Chemicon MAB377), anti-Arc (Sysy #156003), anti-Ki-67 (Abcam ab15580), and anti-Ki-67 APC-conjugated (Invitrogen, clone SolA15). Secondary antibodies for immunohistochemistry were fluorophore-conjugated anti-rat (712- 545-153 and 712-165-153 from Jackson ImmunoResearch), anti-goat (A-11056 from Life Technologies, or 705-166-147 from Jackson ImmunoResearch), anti-guinea pig (Molecular Probe A-11073, and Jackson ImmunoResearch 706-165-148 and 706-175-148 were kind gifts from C.Kintner of the Salk Institute, La Jolla, CA, USA), anti-rabbit (A-10040 or A-21206 from Life Technologies and 711-606-152 from Jackson ImmunoResearch), and anti-mouse (A-21202 from Molecular Probe, 715-166-150 and 715-176-150 from Jackson ImmunoResearch). Key reagents and their usage were as follows: ThioS (Acros Organics 213150250), Hoechst 33258 in a 1% solution of bisbenzimide in water (Sigma-Aldrich B-2883), and paraformaldehyde (PFA) (Sigma P6148).
8
Immunohistochemistry of Mouse Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole mount tissues or cryosections were blocked with 5% donkey serum, 0.1% Triton-X 100, 1% BSA, and 0.02% sodium azide (NaN3) in PBS at pH 7.4 for 1 h at room temperature (RT). Samples were then incubated in primary antibodies overnight at 4 °C. The following primary antibodies were employed: rabbit anti-myosin VIIa (1:200; 25-6790; Proteus BioSciences), goat anti-CD45 (1:100; AF114, R&D systems., Minneapolis, MN, USA), rat anti-F4/80 (1:150, ab6640, Abcam Inc., Cambridge, MA, USA), rat anti-Ly-6G/C (1:100: ab2557, Abcam Inc, USA). The specimens were incubated with secondary antibodies diluted in 0.1% Triton-X 100, 0.1% BSA and 0.02% NaN3 in PBS for 1 h at RT. The secondary antibodies were conjugated with Alexa Fluor 488, 546, and 647 (1:500; A11055, A10040, and A31571, Life Technologies, Carlsbad, CA). After washing with PBS, specimens were mounted in ProLong® Gold Antifade Reagent with DAPI (Cell signaling, #8961 Danvers, MA 01923) and placed under a cover slip. Images were captured using a LSM700 confocal microscope (Zeiss, Germany) at 10X magnification.
9
Immunofluorescent Staining of Hepatocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hepatocytes were grown on collagen-coated confocal dishes and fixed with 3.8% paraformaldehyde for 20 min, after which they were washed three times with ice-cold PBS. To permeabilize, cells and nuclei were treated with 0.25% Triton X-100 in PBS for 10 min. After blocking with 3% BSA, 0.25% Triton X-100, and PBS at RT for 1 h, samples were incubated overnight at 4 °C with the indicated antibodies: TRPM2 (Novus, NB110-81601), CD38 (Thermo Fisher Scientific, 14-0381-85), PKCδ (Santa Cruz, sc-8402), Cx43 (Santa Cruz, sc-13558), PLCδ1 (Santa Cruz, sc-365811), PLCδ3 (Santa Cruz, sc-514912), or Lamin B1 (Santa Cruz, sc-6216). Alexa Fluor-conjugated secondary antibodies (546 donkey anti-rabbit antibody, Thermo Fisher Scientific, A10040; 555 donkey anti-goat antibody, Thermo Fisher Scientific, A-21432; 488 donkey anti-rat antibody, Thermo Fisher Scientific, A-21208; 488 donkey anti-mouse antibody, Thermo Fisher Scientific, A-21202; or 488 donkey anti-rabbit antibody, Thermo Fisher Scientific, A-21206) were incubated at 1:500 dilutions in the presence of 1% BSA at RT for 1 h. The nuclei were stained with DAPI (Thermo Fisher Scientific, 62248). Cells and nuclei were visualized with a Zeiss LSM510 Axiovert 200 M laser-scanning confocal microscope.
10
Visualizing MEF2C and UBE3A Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression plasmids expressing human MEF2C and UBE3A and respective negative control plasmids were obtained from Sino Biologicals (MEF2C-HA: HG12320-CY, UBE3A-Myc:HG11130-CM, pCMV-Myc:CV014 and pCMV-HA:CV013) and used for transient transfection. Transfected HeLa cells were grown on poly-lysine coated coverslips, fixated with 4% paraformaldehyde in PBS for 10 minutes and stained with anti-Myc (M4439, Sigma-Aldrich) and anti-HA (H6908, Sigma-Aldrich) and with Alexa Fluor™ 488 goat anti–mouse and Alexa Fluor™ 488 donkey anti–rabbit antibodies (A11001 and A10040, Thermo Fisher). Nuclei were counterstained with DAPI (Serva). Images were taken with a Zeiss Axio Imager Z2 Apotome microscope with a 63x objective and analyzed in ImageJ.
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!