Cells were seeded into 6-well plates at a density of either 1 × 106 cells per well for analysis on the following day or 0.2 × 106 cells per well if they were to be first subjected to siRNA transfection (for 48 hr). Cells were γ-irradiated, allowed to recover for indicated time periods and then collected and fixed overnight in ice-cold 70% ethanol. Fixed cells were permeabilized with 0.25% Triton-X100 in 1X phosphate-buffered saline (PBS) on ice for 15 min, stained for 2 hr with rabbit anti-phospho-histone H3 (Ser10) Ab (#9701, Cell Signaling) in PBS containing 1% bovine serum albumin (BSA), followed by staining with Fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) in PBS/1% BSA for 30 min. Phospho-H3 positive cells were quantified by fluorescence-activated cell sorting (FACS) on a Cytomics FC-500 flow cytometer (Beckman-Coulter) with laser excitation at 488 nm. Prior to FACS analysis, propidium iodide (PI) (Sigma, P4170) was added to a final concentration of 10 μg/mL to stain DNA. For each sample, 1.5 × 104 events were scored.
Affinipure goat anti rabbit igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
AffiniPure goat anti-rabbit IgG is a laboratory reagent used for the detection and quantification of rabbit immunoglobulin G (IgG) in various experimental and diagnostic applications. It is a highly purified, affinity-isolated antibody preparation that reacts specifically with the heavy and light chains of rabbit IgG.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti phospho histone h3 ser10 ab, by Cell Signaling Technology (2 mentions)
Cytomics fc500 flow cytometer, by Beckman Coulter (2 mentions)
Mouse anti flag clone m2, by Merck Group (2 mentions)
Hrp linked sheep anti mouse igg, by GE Healthcare (2 mentions)
Mouse anti 53bp1, by BD (2 mentions)
Mouse anti γ h2ax clone jbw301, by Merck Group (2 mentions)
Rabbit anti ubiquitin z0458, by Agilent Technologies (2 mentions)
Rabbit anti brca1, by Merck Group (2 mentions)
Alexa fluor 488 goat anti mouse and anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 goat anti mouse and anti rabbit, by Thermo Fisher Scientific (2 mentions)
Rabbit anti gst sc 459, by Santa Cruz Biotechnology (2 mentions)
Mouse anti mbp, by New England Biolabs (2 mentions)
Mouse anti p53 sc 126, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti 53bp1, by Fortis Life Sciences (2 mentions)
Rabbit anti flag, by Cell Signaling Technology (2 mentions)
Mini trans blot cell system, by Bio-Rad (2 mentions)
Mini protean precast gel, by Bio-Rad (2 mentions)
Ab13533, by Abcam (2 mentions)
Trans blot turbo transfer system, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
28 protocols using affinipure goat anti rabbit igg
1
Quantifying Phospho-histone H3 in Irradiated Cells
2
Molecular Profiling of Therapeutic Targets
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Primary antibodies against caspase-3 (9665, CST), caspase-7 (ab32522, Abcam), CCT4 (ab205013, Abcam), GAPDH (97166, CST), PARP (9532, CST), p-STAT1 (Tyr701) (7649, CST), p-STAT2 (Tyr690) (4441, CST), p-STAT3 (Ser727) (9134, CST), p-STAT3 (Tyr705) (9145, CST), p-STAT5 (Tyr694) (4322, CST), p-STAT6 (Tyr641) (9361, CST), STAT1 (9172, CST), STAT3 (12640, CST), STAT5 (94205, CST), STAT6 (5397, CST), XIAP (2045, CST), LC3 (12741, CST), β-catenin (8480, CST), N-cadherin (13116, CST), β-actin (3700, CST), and secondary goat anti-mouse HRP-IgG (7076, CST) and goat anti-rabbit HRP-IgG (7074, CST) antibodies were used for immunoblotting. Primary antibodies against KI67 (9449, CST), CD3 (ab135372, Abcam), CD68 (76437, CST), CD56 (99746, CST), and secondary Fluorescein AffiniPure goat anti-rabbit IgG (111-095-144, Jackson) and Cy3 AffiniPure goat anti-rabbit IgG (111-165-144, Jackson) antibodies were used for immunofluorescence. Antibodies against p-STAT3 (Tyr705) (9145, CST), STAT3 (9139, CST), cleaved caspase-3 (Asp175) (9661, CST), and CCT4 (ab129072, Abcam) were used for immunohistochemistry.
3
Antibodies Used in DNA Damage Assays
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We employed the following antibodies: rabbit anti-53BP1 (A300-273A, Bethyl), mouse anti-γ-H2AX (clone JBW301, Millipore), mouse anti-53BP1 (#612523, BD Biosciences), rabbit anti-GST (sc-459, Santa Cruz), a mouse anti-HA (F-7, sc-7392, SantaCruz or clone 12CA5, gift from M. Tyers, University of Montréal), mouse anti-MBP (E8032S, NEB), mouse anti-Flag (clone M2, Sigma), rabbit anti-Flag (#2368, Cell Signaling), mouse anti-tubulin (clone DM1A, Calbiochem), mouse anti-p53 (sc-126, Santa Cruz), rabbit anti-ubiquitin (Z0458, DAKO), rabbit anti-BRCA1 (#07-434, Millipore or home-made antibody7 (link)). Goat anti-GFP (gift from L. Pelletier, Lunenfeld-Tanenbaum Research Institute), HRP-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch), HRP-linked sheep anti-mouse IgG (NA931, GE Healthcare). Alexa Fluor 488 goat anti-mouse and anti-rabbit IgG, Alexa Fluor 555 goat anti-mouse and anti-rabbit (MolecularProbes).
4
Antibodies Used in DNA Damage Assays
5
Western Blot Analysis of SOD2 Protein
6
Immunostaining for Autophagy Markers
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Mouse anti-DRD2 antibody (1:100; Santa Cruz) was used for the identification of the DRD2-containing neurons. FITC-conjugated AffiniPure goat anti-rabbit IgG (1:200, Jackson ImmunoResearch Laboratories, West Grove, USA) and rhodamin-conjugated AffiniPure goat anti-mouse IgG (1:200, Jackson) were used to recognize rabbit anti-LC3B antibody (1:50; Sigma) and mouse anti- beclin-1(BECN1) antibody (1:50; BD), respectively. DAPI (Sigma) was used for nuclear labeling. Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) solution containing FITC-dUTP (Roche, Basel Schweiz, Switzerland) was used for the detection of DNA fragmentation. Autophagic cell death was identified by the presence of TUNEL (+) BECN1 immunoreactive cells with intact nuclei [23 (link)]. The fields selected for quantification are around the lesion site resided in striatum as shown in panel (C) of S2 Fig S2 Fig
7
ACE2 Expression in Mouse Brain
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For Western Blotting (WB), 6–10 weeks old wild type and ACE2 KO animals were used. Mouse brains were dissected and stored at −80°C. Whole brain lysates were prepared in RIPA buffer supplemented with cOmplete protease inhibitor (Roche Cat # 04693116001). Protein estimation was performed using Pierce BCA protein assay kit (Thermofisher Cat # 23225). Fifteen μg of the sample was loaded in each well of 12% acrylamide gel and SDS–polyacrylamide gel electrophoresis (PAGE) was performed. The proteins were then transferred to Immobilon-P PVDF membranes (Millipore Cat # IPVH00010). Blocking was performed with 5% milk/Tris-buffered saline–Tween 20 for 1 h at room temperature. The membranes were probed with primary anti-ACE2 antibody (Abcam Cat # 15348) and anti-GAPDH (Sigma Cat # G9545) at 1:1000 and 1:5000 dilutions, respectively at 4° C for 16 h. The secondary antibody used was peroxidase-conjugated AffiniPure Goat anti-rabbit IgG (Jackson ImmunoResearch Cat # 111–035-003) at 1:5000 dilution for 1 h at room temperature. Bound antibody was detected using Clarity ECL Western Blotting Substrate (BioRad Cat # 1705061) with the image digitally captured using an ImageQuant LAS 4000 imager.
8
Quantification of Prostatic Inflammation and Protein Expression
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Prostatic inflammatory infiltrates were graded based on a histopathological classification system (Supplementary Table 1
The protein expression intensities were identified using IHC staining. After being deparaffinized, rehydrated, antigen retrieved, and blocked, prostate tissue sections were stained with primary antibodies: TLR2 (1:400; NB100-56720, Novus Biologicals Inc., Littleton, CO, USA), TLR10 (1:500; PA5-20054, Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA), high mobility group box 1 (HMGB1; 1:2000; ab18256, Abcam Inc., Cambridge, MA, USA), and biotinylated secondary antibody: AffiniPure Goat anti-Rabbit IgG (Jackson ImmunoResearch Inc., West Grove, PA, USA). Then, sections were dehydrated and mounted with neutral glue. The mean depth of the brown color (three levels) under the microscope (Olympus, Tokyo, Japan) in four random quadrants was regarded as the intensity of relevant protein expression.
The protein expression intensities were identified using IHC staining. After being deparaffinized, rehydrated, antigen retrieved, and blocked, prostate tissue sections were stained with primary antibodies: TLR2 (1:400; NB100-56720, Novus Biologicals Inc., Littleton, CO, USA), TLR10 (1:500; PA5-20054, Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA), high mobility group box 1 (HMGB1; 1:2000; ab18256, Abcam Inc., Cambridge, MA, USA), and biotinylated secondary antibody: AffiniPure Goat anti-Rabbit IgG (Jackson ImmunoResearch Inc., West Grove, PA, USA). Then, sections were dehydrated and mounted with neutral glue. The mean depth of the brown color (three levels) under the microscope (Olympus, Tokyo, Japan) in four random quadrants was regarded as the intensity of relevant protein expression.
9
Western Blot Analysis of TDG Protein
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SRA01/04 cells were collected and lysed in RIPA containing phenylmethanesulphonyl fluoride (Beytime, Shanghai, China) at 4°C for 30 minutes. Protein extracts were separated on 10% SDS‐PAGE gels and then transferred onto PVDF membranes (Thermo Fisher Scientific, Pittsburgh, PA). After blocking in TBST with 5% non‐fat milk for 2 hours at 37°C, membranes were probed with the primary rabbit monoclonal antibodies against TDG (1:1,000 dilution, Abcam) and GADPH (1:1,000 dilution, Abclonal) overnight at 4°C. After incubation with secondary peroxidase‐conjugated AffiniPure Goat Anti‐rabbit IgG (1:8,000 dilution, Jackson ImmunoResearch), the membranes were exposed to light film (BioMax MR; Kodak, Rochester, NY). The grey value of each protein band was measured, and the TDG protein expression was normalized to GAPDH levels.
10
Western Blot Analysis of iNOS and Arg-1
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Skin samples were lysed with RIPA buffer (BOSTER) and PMSF (BOSTER) to extract protein. The concentration of protein was determined with a BCA protein assay kit (BOSTER). An equivalent amount of protein was loaded onto 10% SDS-PAGE gels (BOSTER), electrophoretically treated and then transferred to PVDF membranes (Millipore). The membranes were incubated with 5% nonfat milk in TBS-T for 2 h at room temperature, and then incubated with primary antibodies at 4 °C overnight. The membranes were incubated with secondary antibodies for 2 h at room temperature, and the protein bands were visualized using ECL reagents. Finally, the bands were photographed using gel image analysis system (Tanon Science & Technology Co., Shanghai, China) and the intensity of the bands was quantified in Gel-Pro analyzer software (Media Cybernetics). The details of the primary and secondary antibodies are as follows: anti-iNOS (1:1000, Thermo Fisher Scientific), anti-Arg-1 (1:500, BOSTER), and horseradish peroxidase-conjugated AffiniPure goat anti-rabbit IgG (1:2000, Jackson ImmunoResearch).
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