The U-87 MG and SK-BR-3 cells were trypsinized, washed three times with PBS, and incubated for 1 h with (ZHER2:4)2DCS-FITC at the concentrations of 0.03, 0.3, and 3.0 μM. As a control, the cells were incubated for 1 h with mouse monoclonal anti HER-2 antibody (AbD Serotec, Bio-Rad, Hercules, CA, USA), washed three times with PBS, and again incubated for 1 h with FITC-conjugated donkey anti-mouse antibody (Jackson Immuno Research, West Grove, PA, USA). Cells were washed three times with PBS and fixed with 1% paraformaldehyde, following their analysis on a FACSDiva instrument (BD Biosciences, Franklin Lakes, NJ, USA). The obtained results were analyzed using WinMidi software (Purdue University, West Lafayette, IN, USA).
Fitc conjugated donkey anti mouse antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
FITC-conjugated donkey anti-mouse antibody is a secondary antibody that binds to mouse primary antibodies. The FITC fluorescent label allows for the detection and visualization of mouse antibodies in various immunoassays and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Cy3 conjugated donkey anti rabbit antibody, by Jackson ImmunoResearch (2 mentions)
Facsdiva, by BD (1 mentions)
Rabbit anti 53bp1, by Cell Signaling Technology (1 mentions)
Vectashield medium, by Vector Laboratories (1 mentions)
Anti rage antibody, by Abcam (1 mentions)
Vectashield dapi, by Vector Laboratories (1 mentions)
Tcs sp8, by Leica (1 mentions)
Ix70 fluorescence microscope, by Olympus (1 mentions)
Ab11267, by Abcam (1 mentions)
Cy3 conjugated donkey anti goat antibody, by Jackson ImmunoResearch (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
4 protocols using fitc conjugated donkey anti mouse antibody
1
HER2 Expression Analysis in Cancer Cells
2
Quantification of DNA Double-Strand Breaks
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DNA DSBs were quantified in spatially (three-dimensionally = 3D) fixed cells by the means of high-resolution ICM detection of co-localized γH2AX and 53BP1 repair foci as described earlier [16 (link),20 (link)]. Briefly, cells were fixed with 4% paraformaldehyde (10 min, at room temperature RT) prior to irradiation (0 min PI, non-irradiated controls) and in several time points PI covering a long (48 h) PI period (5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h and 48 h PI). Cells were permeabilized in 0.2% Triton X-100/PBS (15 min, RT) and immunoassayed with mouse antiphospho-H2AX (serine 139) (Merck, Darmstadt, Germany, cat. no.: 05-636) and rabbit anti-53BP1 (Cell Signaling Technology, Danvers, MA, USA, cat. no.: 4937) primary antibodies to simultaneously detect the γH2AX and 53BP1. Antiphospho-H2AX antibody was visualized with the secondary FITC-conjugated donkey anti-mouse antibody and anti-53BP1 antibody with Cy3-conjugated donkey anti-rabbit antibody (both Jackson Laboratory, West Grove, PA, USA, cat. no.: 715-095-150 and 711-165-152). Chromatin was counterstained with 1 μM TO-PRO-3 (Molecular Probes, Eugene, OR, USA) prepared in 2× saline sodium citrate (SSC). After brief washing in 2× SSC, Vectashield medium (Vector Laboratories, Burlington, Ontario, Canada) was used for sample mounting.
3
Immunofluorescence analysis of RAGE and MAPK
4
Immunofluorescence Analysis of Trigeminal Ganglia
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For immunofluorescence analysis, rats were deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and transcardially perfused them with 100 ml of heparinized normal saline, followed by 500 ml freshly prepared fixative containing 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The TG sections were permeabilized with 50% ethanol for 30 min, blocked with 10% normal donkey serum (NDS) for 30 min, and incubated overnight in the primary antibodies [goat polyclonal antibody to GRP78 (sc1050, 1:200, Santa Cruz Biothechnology), rabbit monoclonal antibody to p-eIF2α (#3597, 1:500, Cell Signaling) and the neuronal marker, microtubule associated protein (MAP2; #ab11267, 1:1,000, Abcam)]. TG sections were washed with 0.01 M phosphate buffered saline (PBS, pH 7.4) and incubated with 2% NDS for 30 min, and incubated with secondary antibodies [Cy3-conjugated-donkey anti-goat antibody, Cy3-conjugated-donkey anti-rabbit antibody and FITC-conjugated-donkey anti-mouse antibody (1:200 in PBS; Jackson Immunoresearch)] for 3 h. Finally, sections were rinsed with PBS, mounted on slides, and coverslipped with Vectashield (Vector, Burlingame, CA) solution. Microscopic observation was performed with an Exi digital camera (Q-Imaging Inc, Surrey, CA) attached to a Zeiss Axioplan 2 conventional fluorescence microscope (Carl Zeiss Inc, Jena, Germany).
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