In conjunction with the cryopreservation, 1 million cells were collected and used for flow cytometry analysis. In short, cells were fixed in paraformaldehyde (HistoLab products AB), resuspended in methanol (−20°C) (cat. 311415, Sigma-Aldrich) and stained for cardiac troponin T using Anti-cardiac Troponin T antibody (ab45932, Abcam) as primary antibody, diluted 1:500, and Alexa flour 488 Fab2 goat anti rabbit IgG antibody (ab150077, Invitrogen), diluted 1:1000, as secondary antibody. The analysis was performed using a Guava® easyCyte HT Sampling Flow Cytometer (Merck Millipore).
Ab150077
Manufactured by Thermo Fisher Scientific
Sourced in Japan, France
The Ab150077 is a lab equipment product from Thermo Fisher Scientific. It is a compact, versatile device designed for use in various laboratory applications. The core function of the Ab150077 is to perform precise measurements and analyses. Detailed specifications and intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab150116, by Thermo Fisher Scientific (2 mentions)
Ab81298, by Abcam (2 mentions)
Ab45932, by Abcam (1 mentions)
Guava easycyte ht sampling flow cytometer, by Merck Group (1 mentions)
Ab31895, by Abcam (1 mentions)
Ab13552, by Abcam (1 mentions)
Faramount aqueous mounting medium, by Agilent Technologies (1 mentions)
700 ax10 laser scanning microscope, by Zeiss (1 mentions)
Ab150115, by Abcam (1 mentions)
Ab134932, by Abcam (1 mentions)
Ab33775, by Abcam (1 mentions)
Ab20034, by Abcam (1 mentions)
Ab32353, by Abcam (1 mentions)
Ab7817, by Abcam (1 mentions)
P36931, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ix51 fluorescence microscope, by Olympus (1 mentions)
Ab177480, by Abcam (1 mentions)
Eclipse ci l microscope, by Nikon (1 mentions)
11 protocols using ab150077
1
Flow Cytometry for Cardiac Troponin T
2
Double-Immunofluorescence of TRPV1 and PSD95
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For double-immunofluorescence of TRPV1 and PSD95 (a marker for post synaptic mechanisms), free-floating sections containing PFC were washed in PBS and then preincubated in PBS containing 5% normal goat serum for 3–4 h at RT. After preincubation, sections were incubated with polyclonal rabbit anti-TRPV1 antibody (ab31895, Abcam, dilution: 1:500), and mouse monoclonal antibody anti-PSD95 (ab13552; Abcam; 1:500) for 24 h at 4°C, after which they were rinsed 3–5 times in PBS, the sections were then incubated for 1–2 h at RT in goat anti-rabbit IgG Alexa Fluor 488 (ab150077; Invitrogen; 1:1500) and goat anti mouse IgG Alexa Fluor 647 (ab150115; Abcam; 1:1500) dissolved in blocking solution. Following the final rinsing, the slices were wet mounted onto subbed slides and subsequently dried and cover-slipped with Dako Faramount Aqueous Mounting Medium (Dako; S3025). For fluorescence imaging, tissues were visualized under ab epifluorescence IX81 microscope (Olympus), and for confocal imaging a 700-AX10 laser scanning microscope (Carl Zeiss) was used.
3
Immunofluorescent Analysis of MMP-9 in NIP
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To investigate MMPs’ distribution in NIP, we performed immunofluorescence staining on NIP tissue sections. After antigen retrieval and antigen blocking. We co-stained MMP-9 and P63 (1:400, Abcam, ab32353) to check the MMP-9 localization and its relationship with the basement membrane of the NIP epithelium. Double staining of alpha-smooth muscle actin (1:200, Abcam, ab7817) and P63 was performed to detect if the finger-like projections contain fibroblasts and capillaries. Co-staining of MMP-9 with neutrophil elastase (1:1000 clone NP57 Dako, Glostrup, Denmark), anti-mast cell antibody (1:200, Abcam, ab134932), ECP (1:200, Abcam, ab1907715), CD4 (1:200, Abcam, ab33775), FOXP3 (1:200, Abcam, ab20034), were performed to determine the cell type that secretes MMP-9. All primary antibodies were incubated at 4°C in the dark overnight, followed by 3 times PBS wash at 5 minutes each. Sections were then incubated with fluorescence secondary antibody (1:500, Abcam, ab150077, ab150116), mounted with DAPI (P36931, Invitrogen), and viewed with a fluorescence microscope (Olympus, Japan).
4
Quantifying FAK Phosphorylation in Adhered HeLa Cells
5
Immunofluorescence Analysis of SOCS3 and NeuN
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The brain tissues were fixed in 10% formalin solution. Paraffin was used to encapsulate the chips, and then 3-μm-thick slices were prepared. The antigen was repaired with sodium citrate buffer in a water bath at 96°C. After washing with PBS and 3% H2O2 for 5 min, the sections were blocked with 10% normal goat serum. To measure the levels of SOCS3 and NeuN, tissue sections were incubated with these primary antibodies at 4°C overnight. After being rinsed with PBS, the sections were incubated with goat polyclonal secondary antibody to rabbit IgG (Alexa Fluor 488, Invitrogen, ab150077) or goat polyclonal secondary antibody to mouse IgG (Alexa Fluor 594, Invitrogen, ab150116) for 2 h at room temperature. After PBS rinse, the sections were incubated with 1 μg/mL DAPI (Sigma) for 10 min, and images were acquired using Olympus IX51 fluorescence microscope and cellSens software.
6
Immunofluorescence Staining of Mouse and Human Skin Tissues
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Paraffin‐embedded mouse cutaneous back tissue was sectioned (4 μm thickness) and stained with hematoxylin and eosin. For IF staining, human ORS cells cultured in FiTC‐P5, sectioned paraffin‐embedded human HFs (7 μm thickness), and sectioned paraffin‐embedded mouse cutaneous dorsal tissue or human skin tissue (5 μm thickness) were incubated at 4°C overnight with the following primary antibodies diluted in the diluent reagent (Invitrogen): phospho‐AMPK (#2535, 1:100; Cell Signaling), Ki‐67 (#M7240, 1:200; Dako), versican (#ab177480, 1:200; Abcam), or AdipoR1 antibody (#ab126611, 1:200; Abcam). After three washes with PBS, the slides were incubated with the secondary Alexa Fluor 488‐labeled anti‐mouse or rabbit IgG antibody (#ab150077, #ab150113, 1:200; Invitrogen), or Alexa Fluor 594‐labeled anti‐rabbit IgG antibody (#a11012, 1:200; Invitrogen) at 25°C for 1 h. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI; #D1306, 1:1,000; Invitrogen). Microscopic observations were performed with a Nikon Eclipse Ci‐L microscope (Nikon). IF intensity was observed and recorded with a Nikon Eclipse Ni‐E fluorescence microscope (Nikon).
7
Multimodal Characterization of Brain
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Rats were sacrificed with transcardiac perfusion with phosphate-buffered saline (PBS), followed by 4% (wt/vol) paraformaldehyde (PFA). Following perfusion, brains were left in 4% PFA for 24 h and then moved to a 30% sucrose solution (wt/vol) in PBS for 2 to 3 days. For chronic electrophysiology, single-cooling dose and acute experiments, a vibratome was used to section the brain into 50 μm coronal or 40 μm sagittal slices, respectively. Coronal slices were stained with NISSL and sagittal slices series were alternated with NISSL or immunostained with a primary antibody against GFP (A-6455, Invitrogen) and a secondary antibody conjugated with AlexaFluor 488 (ab150077), and finally, incubated in DAPI. Images were acquired with a stereoscope (Lumar V12, Zeiss) or a slide scanner (Axio Scan Z1, Zeiss). For animals subjected to bidirectional chronic temperature manipulations, a 1 T MR scanner (ICON, Brucker) was used to collect MRI data. A T 2 -weighted structural image of the brains was collected using a Rapid Imaging with Refocused Echoes (RARE) pulse sequence. The sequence used had a repetition time of 2800 ms, echo time of 90 ms and a RARE factor of 12. The field of view was set to 28 x 15 x 20 mm 2 , the spatial resolution of the images was 150 x 150 x 150 μm 3 or 80 x 80 x 80 μm 3 and a matrix of 187 x 100 x 133 voxels was acquired after 8 averages during a 7-hour scanning.
8
Quantifying FAK Phosphorylation in Adhered HeLa Cells
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HeLa cells were incubated for 90 minutes on TGT surfaces in full culture medium. Then, medium was removed and replaced with 4% (v/v) formaldehyde in PBS and incubated for 15 minutes to allow fixation. Special care was taken to reduce the force fluid shear flow during wash steps so as not to dissociate adhered cells from the surface. Formaldehyde solution was then removed, surfaces were washed with three well volumes of PBS then incubated with 0.1% (v/v) Triton-X in PBS, washed again, then incubated in 100 mg/ml BSA for 1 hr. Cells were then incubated in 1 μg/ml Anti-FAK(phospho Y397) antibody EP2160Y in PBS, rabbit monoclonal (abcam ab81298) for 12 hr at 4°C. Cells were then washed with PBS and stained with 3 μg/ml goat anti-rabbit IgG Alexa Fluor-488(abcam ab150077) in PBS, and 50 μL nuc-blue (Invitrogen R3760)(to confirm cell presence via nuclear stain) in PBS for 1 hr then imaged.
9
Western Blot Analysis of ROCK1 and GAPDH
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The protein samples were obtained using RIPA lysis buffer. Proteins were separated through a 10% SDS-PAGE and incubated with 5% non-fat milk in PVDF membranes at room temperature. Next, we incubated the membranes overnight at 4°C with mouse anti-ROCK1 (1:1,000, rabbit polyclonal antibody, ab97592), anti-GAPDH (1:1,000, rabbit polyclonal antibody, ab9485), which were subsequently incubated with goat anti-mouse secondary antibodies. Then, goat anti-rabbit IgG H&L secondary antibodies (1:1,000, goat polyclonal second antibody, ab150077) protein levels were measured using enhanced chemiluminescence (ECL; Pierce; Thermo Fisher Scientific, Inc.).
10
Detecting Radiation-induced Ecto-CALR in Cancer Cells
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NBTXR3 was added overnight to HCT116, 42-MG-BA and CT26.WT cells before delivering the radiation dose. NBTXR3 concentration, irradiation dose, irradiation source, and the number of individual experiments for each cell line are reported in Additional file 1 : Table S2. For analysis of cell-surface exposure of calreticulin (Ecto-CALR), cells were harvested using Accutase (#A6964, Sigma-Aldrich, France) and fixed in 0.25% paraformaldehyde (#11,586,711, Thermo-Fisher, France) 24 h after irradiation. A rabbit anti-calreticulin primary antibody (#Ab4, Abcam, France) was added to cells and visualized using an anti-rabbit Alexa 488 (#Ab150077, ThermoFisher, France). Cells were counter-stained with Live/Dead fixable Far Red Dead Cell stain kit (#L34974, Thermo-Fisher, France) to exclude dead cells and Ecto-CALR was measured by cytofluorometry (Accuri C6+, Becton Dickinson, France).
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