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.
Cy3 conjugated donkey anti rabbit antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Cy3-conjugated donkey anti-rabbit antibody is a secondary antibody that binds to rabbit primary antibodies. The Cy3 fluorescent dye is conjugated to the antibody, allowing for detection and visualization of target proteins in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (3 mentions)
Fitc conjugated donkey anti mouse antibody, by Jackson ImmunoResearch (2 mentions)
Primary rat anti s100a8, by Thermo Fisher Scientific (2 mentions)
Alexafluor 488 conjugated donkey anti rat antibody, by Jackson ImmunoResearch (2 mentions)
Cryojane tape transfer system, by Leica (2 mentions)
Cryostat, by Leica (2 mentions)
Dabco, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Rabbit anti 53bp1, by Cell Signaling Technology (1 mentions)
Vectashield medium, by Vector Laboratories (1 mentions)
Ab11267, by Abcam (1 mentions)
Cy3 conjugated donkey anti goat antibody, by Jackson ImmunoResearch (1 mentions)
N9528, by Merck Group (1 mentions)
Anti phosphorylated p38 mapk, by Cell Signaling Technology (1 mentions)
Fluoview fv1000 bx2 upright confocal, by Olympus (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Egg albumin, by Merck Group (1 mentions)
Menzel glass, by Thermo Fisher Scientific (1 mentions)
Gelatin, by Merck Group (1 mentions)
Streptavidin alexa488, by Jackson ImmunoResearch (1 mentions)
9 protocols using cy3 conjugated donkey anti rabbit antibody
1
Quantification of DNA Double-Strand Breaks
2
Immunofluorescence Staining of Spleen Cryosections
3
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).
4
Immunohistochemical Analysis of Hippocampal NPY
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Sections were fixed in 4% paraformaldehyde for 20 min. Sections were washed three times in KPBS for 10 min and preincubated in 10% Donkey serum, 1% Triton X-100 in KPBS for 1 h. After adding rabbit anti-NPY antibody (1:500, N9528, Sigma-Aldrich, DK) in 5% Donkey serum, 0.25% Triton X-100 in KPBS, sections were incubated overnight at 4 °C. Sections were washed 3 × 10 min in tKPBS, incubated for 2 h at RT with Cy3-conjugated Donkey anti-rabbit antibody (1:200, Jackson Immunoresearch USA) in 1% Donkey serum, 0.25% Triton X-100 in KPBS and washed 1 × 10 min in tKPBS and 2 × 10 min in KPBS. The slides were then mounted with DABCO (Sigma-Aldrich) and digitized images were obtained using Olympus BX61 microscope and CellSens software. Average fluorescence intensity of NPY immunoreactivity in hippocampus was evaluated using ImageJ. The experimenter performing the evaluation was unaware of the specific treatment given to individual slices.
5
Immunofluorescence Staining of Spleen Cryosections
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Spleens were fixed in 1% formaldehyde (StatLab, McKinney, TX) for 4–8 h, rehydrated in 30% sucrose solution for 72 h, and snap frozen in OCT (Sakura Finetek, Japan). Single-cell-thick (5 μm) spleen cryosections were obtained using a Leica Cryostat and the CryoJane tape transfer system (Leica Microsystems, Wetzlar, Germany). For immunofluorescent staining, slides were rehydrated in PBS for 10 min followed by rinsing in PBST (PBS + 0.1% Tween20); blocking was performed with PBS + 10% donkey serum for 20 min; the diluted primary rat anti-S100a8 (Thermo Fisher Scientific #335806) and rabbit anti-IFIT1 (Abcam, Cambridge, UK #ab236256) antibodies were added and incubated for 1 h at RT. After 3x washes with PBST, AlexaFluor 488-conjugated donkey anti-rat antibody (Jackson ImmunoResearch, West Grove, PA #141697) and Cy™3-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch #143460) were added and incubated for 30 min at RT. Slides were washed 3x with PBST and then stained with DAPI (0.5 μM) for 3 min. Slides were rinsed in PBS and were covered with mounting solution (Vectashield, Vector Laboratories, Burlingame, CA).
6
Quantifying Phosphorylated p38 MAPK in Spinal Cord Dorsal Horn
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Freshly prepared 10 μm spinal cord sections were fixed with 4% paraformaldehyde and blocked with 3% donkey serum. Sections were then permeabilized with 0.01% Triton X-100 (all from Sigma-Aldrich) and then incubated with primary antibodies, anti-phosphorylated p38 MAPK (1:50) (1:100, Cell Signaling Technology). The bound antibodies were detected by incubation with Cy3 conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch) and counterstained with DAPI (Thermo Fisher Scientific) to detect the nulei. Confocal images from the dorsal horn were collected using the Olympus FluoView FV1000 BX2 Upright Confocal at the University Imaging Centers of University of Minnesota. The average percentage of co-expression of phosphorylated p38 MAPK with cell nuclei throughout the spinal cord dorsal horn (six images acquired from randomly selected areas from superficial to the deep dorsal horn) was quantified with Image J.14 (link)
7
Immunohistochemical Visualization of NPY
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Sections were washed three times in KPBS for 10 min and imbedded in a solution containing 300 g/L egg-albumin (Sigma-Aldrich) and 30 g/L gelatin (Sigma-Aldrich), frozen at −80 °C and sub-sliced on a cryotome at −20 °C (Cellab Nordia AB) to 16 μm thick slices. Slices were mounted on positively charged glasses (+Menzel glass, Thermo Scientific) and stored at −20 °C. These slices were then preincubated in 10% Donkey serum, 0.25% Triton X-100 in KPBS for 1 h. After adding rabbit anti-NPY antibody (1:500, #N9528, Sigma-Aldrich, DK) in 5% Donkey serum, 0.25% Triton X-100 in KPBS, sections were incubated overnight at 4 °C. Sections were washed 3 × 10 min in tKPBS, incubated for 2 h at RT with Cy3-conjugated Donkey anti-rabbit antibody (1:200, Jackson Immunoresearch USA) in 1% Donkey serum, 0.25% Triton X-100 in KPBS and washed 1 × 10 min in tKPBS and 2 × 10 min in KPBS. The slides were mounted with DABCO (Sigma-Aldrich) and digitized images obtained using Olympus BX61 microscope and CellSens software.
8
Visualizing RPL10 Localization in Yeast
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For direct fluorescence experiments, cells were grown to saturation in selective medium containing galactose, then diluted back 20-fold in medium containing 2% glucose and grown for an additional hour to repress the expression of genomic RPL10. Images were captured using a Nikon E800 microscope fitted with a 100x Plan Apo objective and a Photometrics CoolSNAP ES camera controlled by NIS- Elements software. For indirect immunofluorescence, cells were grown in selective medium containing galactose before adding glucose to 2% to repress the expression of wild-type genomic RPL10 for 2h. LMB was added to a final concentration of 0.1 μg/ml for 15 min to block Nmd3 shuttling. Cells were fixed with a 1:9 volume of 37% formaldehyde for 40min, then washed with Ksorb buffer (0.1 M potassium phosphate [pH 6.6], 1.2 M sorbitol). Cells were permeabilized in cold methanol followed by washing in acetone. Anti-Nmd3 antibody [49 ] was diluted 3,000 fold in PBS with 0.1% BSA. Cy3-conjugated donkey anti—rabbit antibody (Jackson ImmunoResearch Laboratories, Inc.) was used at a 300-fold dilution. After antibody application, cells were incubated for 1 min in 1 μg/ml DAPI and mounted in Aqua-Poly/Mount.
9
Immunohistochemical Labeling of Parvalbumin and Microglia in Rat Brain
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Rats were perfused at the end of the fourth week with physiological saline, followed by a 4% paraformaldehyde solution. Details of the procedure are described elsewhere [6] . 80-m-thick coronal cryosections through the brain specimens were collected in 0.1 M phosphate buffer (pH 7.3). All sections along the rostrocaudal aspect of the parvafox nucleus (in general 28 [range: 25-30] sections, spanning a total length of 2.24 [range: 2.0-2.4] mm) were incubated first with a monoclonal primary antibody against PV (PV235, 1:1000, Swant, Marly, Switzerland) for 24-48 h at 4 • C, then with biotinylated anti-mouse IgG (1:200, Vector Laboratories, Burlingame, CA) for 2 h at ambient temperature, and finally with streptavidin-Alexa488 (1:200, Jackson Immunoresearch Laboratory, West Grove, PA) for 3 h at ambient temperature. In addition, every fourth section of the specimens from rats in cohort two was exposed first to rabbit anti-Iba1 (ionized calcium-binding adaptor molecule 1; Wako Pure Chemicals, Osaka, Japan [0.25 g/ml]) for 24 h at 4 • C and then to Cy3-conjugated donkey anti-rabbit antibody (Jackson Immunoresearch Laboratory [1:200] ) for 2 h at ambient temperature.
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