BMNCs in IR− group and IR−+SVP-B5 group were collected 2 h after injection of saline or SVP-B5, IR+ group and IR++SVP-B5 group were treated as described above and B MNCS were collected at 7d and 14d after TBI. The BMNCs were fixed with 100% ethanol for 5 min. The samples were centrifuged and the cells were incubated with 0.1% PBS-Tween for 20 min and centrifuged. After washing with 0.1% PBS-Tween, a mixture of 10% goat serum/PBS/0.3 M glycine was added and the sample was mixed well and placed at 22 °C for 30 min. The non-specific reaction between proteins was blocked. The sample was washed and a sample of 100 μl γH2AX antibody (Abcam, USA)diluted 1:500 was added, and incubated at 22 °C for 30 min. The sample was washed and centrifuged and the supernatant was discarded. The pellets were treated with 100 μl secondary antibody (DyLight® 488 goat anti-mouse IgG) (H+L) (Abcam, USA) diluted 1:500 and incubated at 22 °C for 30 min, washed and centrifuged. Subsequently, 0.5 ml 0.1% PBS-Tween was added, and the γH2AX level was detected by flow cytometry at the FITC wavelength.
Dylight 488 goat anti mouse igg h l
Manufactured by Abcam
Sourced in United States, United Kingdom, Italy
DyLight® 488 goat anti-mouse IgG (H+L) is a secondary antibody conjugated with the DyLight® 488 fluorescent dye. It is designed for the detection of mouse immunoglobulins in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
γh2ax antibody, by Abcam (1 mentions)
Fc500 flow cytometer, by Beckman Coulter (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Glycine, by Merck Group (1 mentions)
Ab16066, by Abcam (1 mentions)
Concanavalin a tetramethyl rhodamine conjugate, by Thermo Fisher Scientific (1 mentions)
Ez c1, by Nikon (1 mentions)
Ab92824, by Abcam (1 mentions)
Cellquest 3, by BD (1 mentions)
Facs caliber, by BD (1 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (1 mentions)
Ab18207, by Abcam (1 mentions)
Ab53025, by Abcam (1 mentions)
Ab10062, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
5 protocols using dylight 488 goat anti mouse igg h l
1
Quantifying DNA Damage in Traumatic Brain Injury
2
HIF-1α Expression in PC12 Cells
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PC12 cells were seeded at 4 × 104 cells /mL and incubated for 24, 48, 72 and 96 h. Following incubation, the cells were fixed with 80% ice-cold methanol (Thermo Fisher Scientific, Waltham, MA, USA) for 5 min and the methanol was removed. After permeabilisation with 0.1% PBS/Tween for 20 min, cells were incubated in a blocking solution consisting of 10% Bovine Serum Albumin (BSA) with 0.3 M glycine (Sigma-Aldrich, St. Louis, MO, USA) with primary monoclonal antibodies for HIF-1α (ab16066) (Abcam, Cambridge, UK) at a concentration of 2 µg/1 × 106 cells and incubated for 30 min at 22 °C. The primary antibody-blocking solution was then removed via centrifugation at 300× g for 3 min. The cells were washed with PBS, centrifuged for 3 min at 300× g and then incubated with 500 µL of secondary antibody DyLight 488 goat anti-mouse IgG (H + L) (Abcam, Cambridge, UK) (1/500 dilution) for 30 min at 22 °C. An FC500 flow cytometer (Beckman Coulter, Brea, CA, USA) was used to analyse the samples. At least 10,000 events were collected per sample. Data were analysed using Flowing Software (Turku Centre for Biotechnology, Turku, Finland).
3
Immunofluorescent Staining of SCD1 in Ovarian Cancer
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OVCAR-3 mock and OVCAR SCD1 O.E. tumor cells were stained by anti-SCD1 Ab (clone: CD.E10; ab19862, Abcam). The secondary antibody used was DyLight 488 goat anti-mouse IgG (H+L; ab96879, Abcam) at 1:500 dilution for 1 hour at 4°C and then washed three times with FACS buffer (2% FCS PBS).
4
Mitochondrial Changes in Human Cardiac Cells
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Morphological changes and mitochondrial activity of human cardiac cells were studied through a Confocal Laser Scanning Microscope (EZ-C1-Nikon). Briefly, human cardiac cells were untreated (control) or treated with DOXO alone or combined with DAPA for 24 h. After incubation, cardiomyocytes were fixed in 4% formaldehyde (10 min) and then incubated in 1% BSA/10% normal goat serum/0.3 M glycine in 0.1% PBS-Tween20 for 1 h to permeabilize the cells and block non-specific protein–protein interactions. The cardiomyocytes were then incubated with an anti-Mitochondria antibody (113-1)—BSA and Azide free (Abcam ab92824, Milan, Italy) 5 µg/ml overnight at ±4°C. As a secondary antibody (green), a DyLight® 488 goat anti-mouse IgG (H ± L) (ab96879, Abcam, Milan, Italy) was used at a dilution of 1/250 for 1 h. Membrane staining was obtained using Concanavalin A Tetramethylrhodamine Conjugate (Invitrogen, Life Technology, Milan, Italy) at a final concentration of 100 µg/ml. Through a confocal microscope (C1-Nikon) equipped with EZ-C1 software for data acquisition and 60× oil immersion objective, intracellular mitochondria were imaged through excitation/emission at 488/518 nm and cell membrane through excitation/emission at 555/580 nm (31 (link)).
5
Quantifying Neurogenic Markers in ADMSCs
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On day 1 after the second step of induction, the ADMSCs were subjected to flow cytometry to quantify the proportion of cells with positive neurogenic markers. The cells were fixed using 4% paraformaldehyde at room temperature for 30 min and permeabilized using 0.1% TritonX-100+2% BSA in PBS at 37°C for another 30 min. Then the cells were incubated with primary anti-NSE antibody (1:100, ab53025, Abcam) for one hour, anti-GFAP (1:100, ab10062, Abcam) for 30 min or anti-Tuj-1 (1:100, ab18207, Abcam) for 30 min. For anti NSE and anti-Tuj-1, the secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (1:4000, ab150077, Abcam) for 30 min at 22°C. For anti-GFAP, the secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (1:500, ab96879, Abcam) for 30 min at 22°C. The proportion of cells with active neuronal markers were then analyzed using a FACSCaliber (BD Biosciences, Franklin Lakes, NJ, USA). Data acquisition was done using CellQuest 3.2 software (BD Biosciences). Each test was performed with at least three repeats.
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