Paraffin sections were cut at 4 μm, placed on charged slides, and dried at 60°C for 1 hour. Slides were cooled to room temperature, deparaffinized in three changes of xylene, and rehydrated using graded alcohols. For antigen retrieval, slides were heated in the steamer for either 40 or 60 minutes in citrate buffer (pH 6.0) (Biocare Medical, (Concord, CA) No. CB910), followed by a 20-minute cooldown. Endogenous peroxidase was quenched with aqueous 3% H2O2 for 10 minutes and washed with PBS/T (Tween-20). Slides were loaded on a Dako (X0909, Dako, CA) autostainer, serum-free protein block (Dako No. X0909) was applied for 5 minutes and blown off, and the RRM2 antibody (SC-81850, Santa Cruz Biotechnology (Santa Cruz (Dallas, TX)) was applied at 1:500 dilution for 1 hour. PBS/T was used to wash slides between each reagent application. Dako Mouse Envision (K4007) was applied for 30 minutes, followed by DAB (Dako No. K3468) for 10 minutes. Finally, the slides were removed from the autostainer, counterstained with hematoxylin, dehydrated, cleared, and coverslipped. Isotype-specific nonimmune IgG was used as a control to examine the specificity of RRM2 staining.
X0909
Manufactured by Agilent Technologies
Sourced in United States, Denmark, Canada, Belgium
The X0909 is a laboratory instrument designed for analytical applications. It provides accurate and reliable measurements for various research and testing purposes. The core function of the X0909 is to perform precise analysis and data collection.
Automatically generated - may contain errors
Lab products found in correlation
S2023, by Agilent Technologies (12 mentions)
K3468, by Agilent Technologies (10 mentions)
K4003, by Agilent Technologies (8 mentions)
S2367, by Agilent Technologies (8 mentions)
S3022, by Agilent Technologies (7 mentions)
S1699, by Agilent Technologies (7 mentions)
Envision dual link system hrp, by Agilent Technologies (4 mentions)
Sk 4200, by Vector Laboratories (4 mentions)
Apotome, by Zeiss (4 mentions)
Pk6106, by Vector Laboratories (3 mentions)
K8007, by Agilent Technologies (3 mentions)
Autostainer, by Agilent Technologies (3 mentions)
Mhs32, by Merck Group (3 mentions)
C0563, by Agilent Technologies (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
D9542, by Merck Group (2 mentions)
S3309, by Agilent Technologies (2 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (2 mentions)
Af3975, by Novus Biologicals (2 mentions)
Nbp2 13816, by Novus Biologicals (2 mentions)
112 protocols using x0909
1
Immunohistochemical Analysis of RRM2
2
Immunohistochemical Analysis of γ-H2AX in Tumors
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Tumors were harvested 2 weeks after the beginning of treatments and were formalin-fixed and paraffin-embedded. Four micrometer thick paraffin-embedded tissue sections were cut and immunostained using an automated Ventana Discovery XT staining system (Ventana Medical Systems). Antigen retrieval was performed in cell conditioner 1 and slides were incubated with anti-phospho-histone γ-H2AX antibody (1:250 dilutions in PBS at 37 °C for 60 min, clone JBW301, EMD Millipore, Temecula, CA) on automatic. Then, on the bench, slides were incubated for 20 min with blocking solution (Dako, Agilent, #X0909) followed by incubation with the secondary antibody (1:250 dilution in PBS at RT for 45 min; anti-mouse Cy5, Life Technologies Inc., #A10524). Finally, slides were incubated 15 min at RT with a 0.1% (w/v) solution of Sudan Black in 70% ethanol to quench tissue auto fluorescence. Slides were mounted using ProLong® Gold Antifade Mountant with DAPI (Life Technologies Inc., P-36931). Between each step, except after the blocking steps, slides were washed twice with PBS. Slides were stored at 4 °C and scanned the next day. A negative control slide was done in parallel where PBS replaced the primary antibody.
3
Immunohistochemical analysis of neurodegeneration
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Following target retrieval, human brain sections were blocked with serum-free protein block (Agilent, X0909) for 1 h at room temperature and incubated in primary antibodies against p-S65-Ub (in-house; 1:500) [25 ], PPIF (Abcam, ab110324; 1:500), CTSD (Millipore Sigma, IM03; 1:1000), CSNK1D (Santa Cruz Biotechnology, sc-55553; 1:50), SNCA (Invitrogen, 180215; 1:100), p-S129-SNCA (Wako Chemicals USA, 015–25191; 1:3000), p-S202-MAPT (monoclonal antibody from Peter Davies, Feinstein Institute, CP13; 1:250) or p-S396- and p-S404-MAPT (monoclonal antibody from Peter Davies, PHF-1; 1:500) diluted with antibody diluent with background reducing components (Agilent, S302283) at 4°C overnight in a humidity chamber. The next day sections were incubated with secondary antibodies (Invitrogen, A-11034 and A-11004; 1:1000) and DAPI (Sigma-Aldrich, D9542; 1:1000) at room temperature for 1.5 h. Autofluorescence signal was quenched using 3% Sudan black (SPI Supplies, 02560-AB) in 70% MeOH (Pharmco-Aaper, AAP-1031) for 2 min before coverslipping slides using fluorescence mounting medium. Super-resolution confocal (Airyscan) fluorescence images were taken with a Zeiss observer LSM 880 microscope equipped with 405-, 488-, 543-, and 633-nm lasers (Zeiss, Oberkochen, Germany).
4
Immunohistochemical Analysis of Cellular Markers
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The formalin-fixed and paraffin-embedded tissue blocks were sectioned (2 μm) and subjected to deparaffinization, rehydration and antigen retrieval by PT Link (Agilent, PT10126), at low or high pH as suggested by the primary antibody datasheets used. Endogenous peroxidase was blocked for 10 min with a peroxidase blocking solution (Agilent Dako, S2023) and, successively, nonspecific antibody binding was blocked for 20 min with protein blocking buffer (Agilent Dako, X0909). Tissue sections were immunostained for 1 h at RT with anti-TERF2/TRF2 rabbit polyclonal (1:500), anti-8-OHdG mouse monoclonal (1:800), anti-LC3B rabbit monoclonal (AbCam, EPR21234, 1:100) and then were covered for 30 min at RT with Dako EnVision™ FLEX /HRP (EnVision™ FLEX; Agilent, K8023). The signal was developed by using DAB detection kit (Agilent Dako, GV825), then sections were counterstained with Mayer’s Hematoxylin (Agilent Dako, S3309). Finally, slides were washed, dehydrated with increasing alcohol and xylene and mounted with Eukitt (Sigma-Aldrich, 03989). Immunostaining results were recorded as percentage of positive cells or immunoreactive score (IRS, staining intensity per percentage of positive cells).
5
Immunohistochemical Analysis of Pancreatic Islets
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Immunohistochemistry was carried out as previously described [37 (link)]. Prior to adding the primary antibody, the slides were treated with serum-free protein block (X0909, Agilent Dako). Primary antibodies and dilutions were anti-PAVIRF (1:5000), anti-PRAAHG (1:5000), and anti-CEL (HPA052701, Sigma-Aldrich; 1:100-200). Primary antibody detection was done by MACH3 anti-rabbit probe and HRP-conjugated polymers (Biocare Medical), followed by visualization with 3,3`-diaminobenzidine (Dako). The sections were counterstained with hematoxylin and images acquired by using a Leica DMLB microscope with the ZEN 2011 software. Alternatively, the sections were scanned using (Nano Zoomer XR, Hamamatsu Photonics) and images obtained by Aperio ImageScope (Leica Biosystems). The software QuPath (qupath.github.io ) was used for quantification of INS-positive cells in mouse pancreatic sections.
6
Histochemical Detection of Alkaline Phosphatase
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Four micrometer sections from decalcified, paraffin-embedded bones from the 37% site of the total length of the tibia from the proximal end were used for detection of alkaline phosphatase (ALP)–positive cells. Sections were immunostained using a rabbit polyclonal anti-ALP antibody (ab97384; Abcam, Cambridge, United Kingdom) and a horseradish peroxidase-labeled anti-rabbit antibody using 3,3’-diaminobenzidine as substrate (K4003; Agilent, Santa Clara, CA, USA) according to the manufacturer’s description. Antigen retrieval was performed in citrate buffer (pH = 6.0) overnight at 60°C. Background staining was reduced by incubating the sections in 3% H2O2 diluted in methanol for 20 min. Sections were incubated for 2 h at room temperature with the antibody diluted 1:30 in protein-blocking solution (X0909; Agilent). Nuclei were counterstained with hematoxylin. All washing steps were performed in PBS.
7
Immunohistochemical Analysis of Pancreatic Cancer
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Immunohistochemical analysis was performed on pancreatic cancer specimens, as previously described [28] . Briefly, tissue specimens were fixed in 10% Formalin Neutral Buffer Solution (FUJIFILM Wako Chemicals, 062-01661) and embedded in paraffin wax before sectioning. For epitope retrieval, the sections were heated in 10 mM Citrate Buffer (pH 6.0) at 95 °C for 40 min using a water bath. Peroxidase blocking was performed in 0.3% H 2 O 2 / MeOH for 30 min at room temperature. The sections were preincubated with a serum-free protein block (Agilent Technologies X0909) for 30 min at room temperature, and primary antibodies were added to each slide at 4°C overnight. Secondary antibody staining was performed using EnVision +/ HRP, Rabbit (Agilent Technologies K4003) for 30 min at room temperature. HRP activity was visualized using chromogenic substrate 3, 3'-Diaminobenzidine (DAB). Staining was visualized under a Nikon Eclipse Ni-U microscope (NIKON CORPORATION, Tokyo, Japan). The histochemical intensity was analyzed using the ImageJ software version 1.52a (SciJava software ecosystem's open-source software project) [29, 30] .
8
Quantifying Advanced Glycation End-Products in Cardiomyocytes
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Transversal frozen sections of 10 µm were obtained and immunohistologically stained for AGEs with DAB staining. First, antigen retrieval was performed with citrate buffer (pH = 6). The sections were blocked for 1 h at room temperature with serum-free protein block (X0909, Dako Agilent, Diegem, Belgium). Tissue sections were incubated overnight at 4 °C with a rabbit anti-rat primary antibody for AGEs (1/250, Abcam, ab23722). EnVision™ + Dual Link System-HRP (Dako Agilent, anti-rabbit/anti-mouse, K4061) was applied for 30 min at room temperature. DAB solution was added (Dako Agilent) and sections were counterstained with hematoxylin. Coupes were dehydrated and mounted with DPX mounting medium. Negative controls were included in each staining, in which the staining procedure was performed with omission of the primary antibody. Images were acquired using a Leica MC170 camera connected to a Leica DM2000 LED microscope. The AGEs deposition was quantified with Fiji/ImageJ software (1.53c) in four randomly chosen regions. The AGEs-positive area was normalized to the total cardiomyocyte area and expressed as AGEs’ content in %.
9
Immunofluorescence Assay for IL-1β-Treated Cells
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The human NP cells were plated in 4-well culture slide and incubated for 24 h. The cells were treated with 10 ng/ml IL-1β with or without T-5224, then fixed for 10 min with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS for 8 min, blocked with protein block (X0909, Agilent Technologies, USA) for 1 h, and incubated overnight at 4 °C with primary antibodies. The cells were washed and incubated with anti-rabbit Alexa Fluor and DAPI for 1 h at room temperature for nuclear staining.
10
Immunohistochemistry of PDAC Tissue
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Paraffin embedded zinc-formalin fixed tissue was sectioned and antigen retrieval was performed with citrate pH6.0 buffer
in a pressure cooker. Slides were blocked with serum-free protein block (X0909, Dako) for 1 hour at RT and incubated overnight at
4°C with the following antibodies FOXM1 (AF3975, Novus, 1:100), PRRX1 (NBP2-13816, Novus, 1:50) or GFP (ab13970, abcam,
1:250). Immunohistochemistry was performed using Biotinylated secondary antibodies, ABC kit (PK-6100) and DAB (SK-4100) from
Vector Labs. For immunofluorescence, slides were incubated with Cy-conjugated secondary antibodies (Jackson ImmunoResearch, 1:300)
for 1 hour at RT and nuclei were stained with DAPI. Image were acquired on a Nikon E600 microscope and processed with ImageJ
(1.51w) software. Antibody fidelity was verified by staining murine PDAC tissue in the absence of primary antibody (Supplementary Figure 1A ). Human PDAC tissue was obtained from US Biomax,
Inc. (PA483).
in a pressure cooker. Slides were blocked with serum-free protein block (X0909, Dako) for 1 hour at RT and incubated overnight at
4°C with the following antibodies FOXM1 (AF3975, Novus, 1:100), PRRX1 (NBP2-13816, Novus, 1:50) or GFP (ab13970, abcam,
1:250). Immunohistochemistry was performed using Biotinylated secondary antibodies, ABC kit (PK-6100) and DAB (SK-4100) from
Vector Labs. For immunofluorescence, slides were incubated with Cy-conjugated secondary antibodies (Jackson ImmunoResearch, 1:300)
for 1 hour at RT and nuclei were stained with DAPI. Image were acquired on a Nikon E600 microscope and processed with ImageJ
(1.51w) software. Antibody fidelity was verified by staining murine PDAC tissue in the absence of primary antibody (
Inc. (PA483).
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