X0931

Manufactured by Agilent Technologies

Sourced in Denmark, United States

The X0931 is a high-precision laboratory equipment designed for analytical and measurement applications. It features advanced sensors and data processing capabilities to provide accurate and reliable measurements. The core function of the X0931 is to enable precise quantitative analysis and data collection for research and scientific purposes.

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Lab products found in correlation

Pronase, by Merck Group (3 mentions) Hyaluronidase, by Merck Group (3 mentions) Ii ii6b3, by Developmental Studies Hybridoma Bank (3 mentions) Hk 321 uk, by BioGenex (3 mentions) M7240, by Agilent Technologies (2 mentions) Normal goat serum (ngs), by Merck Group (2 mentions) Hk 325 um, by BioGenex (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Goat anti rat cy3, by Jackson ImmunoResearch (1 mentions) Rat anti cd45 clone 30 f11, by BioLegend (1 mentions) Clone m5 114, by BioLegend (1 mentions) Rat anti f4 80 clone bm8, by BioLegend (1 mentions) Rat anti cd11b clone m1 70, by BioLegend (1 mentions) Rat anti cd68 clone fa 11, by BioLegend (1 mentions) M0851, by Agilent Technologies (1 mentions) Cd11c clone n418, by BioLegend (1 mentions) Immpress anti mouse igg polymer detection kit, by Vector Laboratories (1 mentions) Ab2557, by Abcam (1 mentions) Ab13847, by Abcam (1 mentions) Ab191183, by Abcam (1 mentions)

20 protocols using x0931

Immunostaining of Optically Cleared Tissues

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The following primary antibodies were used for immunostaining of optically cleared tissues: polyclonal rabbit anti‐α‐smooth muscle actin (SMA) (Abcam; ab5694; 1 : 300); and from (BioLegend UK Ltd, London, UK): rat anti‐CD45 clone 30‐F11 (103102; 1 : 300), CD11c clone N418 (117302; 1 : 200) and MHCII I‐A/I‐E clone M5/114.15.2 (107601; 1 : 300). The following macrophage markers were found to be unreliable with the tissue clearing protocols used: rat anti‐F4/80 (clone BM8), rat anti‐CD11b (clone M1/70), rat anti‐CD68 (clone FA‐11) (all from BioLegend) and rat anti‐F4/80 (clone Cl:A3‐1; from AbD Serotec, Kidlington, UK). The following secondary antibodies (all used at 1 : 500) were purchased from Invitrogen: goat anti‐rabbit Alexa Fluor 488 (A11008), goat anti‐rat Alexa Fluor 647 (A21247) and goat anti‐rat Cy3 (A10522); and from (Jackson ImmunoResearch, Ely, UK): goat anti‐Armenian hamster Cy3 (127‐165‐160).
For 2D analysis, SMA expression was detected using a mouse anti‐human SMA primary antibody (clone 1A4, Agilent; M0851) with a peroxidase‐conjugated ImmPRESS anti‐mouse IgG polymer detection kit (Vector Laboratories Ltd; MP‐7402, Peterborough, UK) using standard development with DAB. Mouse IgG1 was used for species‐ and isotype‐matched control (Agilent; X0931).

Hitchcock J.R., Hughes K., Harris O.B, & Watson C.J. (2019). Dynamic architectural interplay between leucocytes and mammary epithelial cells. The Febs Journal, 287(2), 250-266.

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Quantification of Neuroinflammation and Tissue Damage

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Bain sections were incubated overnight with primary antibodies at 4 °C. Primary antibody staining was detected with fluorophore-conjugated secondary antibodies. Images were captured using a NanoZoomer S60 Digital slide scanner (Hamamatsu, Japan). Cell counting (neutrophil and caspase3) and immunostaining area (hemoglobin and IgG) were performed by 2 independent operators blinded to the treatment of the animals by using ImageJ software with thresholds set according to signal intensity. For the evaluation of Hb staining in the infarcted brain, we quantified the percentage of area of Hb immunostaining by using ImageJ software (2 different zones were chosen (1: cortex and 2: striatum) with 5–7 sections analyzed per mice). The following primary antibodies were used: rabbit anti-hemoglobin (1:200, ab191183, Abcam, Paris, France), rat anti-neutrophil (1:200, ab2557, Abcam, Paris, France), Alexa-Fluor 594 Donkey anti-mouse IgG (1:500, X0931, Dako, Agilent Technologies, Santa Clara, CA, USA), and rabbit anti-caspase-3 (1:100, ab13847, Abcam, Paris, France). The following secondary antibodies were used: Alexa-Fluor 488 Donkey anti-Rabbit (1:1000, A-21206, invitrogen Molecular Probes, Invitrogen, Carlsbad, CA, USA) and Alexa-Fluor 594 Donkey anti-Rat (1:500, A-21209, invitrogen Molecular Probes, Invitrogen, Carlsbad, CA, USA).

Couret D., Planesse C., Patche J., Diotel N., Nativel B., Bourane S, & Meilhac O. (2021). Lack of Neuroprotective Effects of High-Density Lipoprotein Therapy in Stroke under Acute Hyperglycemic Conditions. Molecules, 26(21), 6365.

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Histological Analysis of Redifferentiated Chondrocytes

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At day 14 of redifferentiation, the alginate beads of all three donors and six conditions were fixed overnight in 100 mM CaCl₂ in 4% neutral buffered formaldehyde solution and thereafter dehydrated in ethanol.⁴³ The next day, the alginate beads were embedded in paraffin, 5 μm sections were cut and stained with H&E (109249 and 115935, Merck). Immunohistochemistry (IHC) staining for collagen type I and II was performed as described previously.⁴⁴ IHC for aggrecan (mouse monoclonal recombinant antibody, 4 μg/ml; ab3778, Abcam) was performed for collagen type I and II, but with 60 instead of 30 min of 1 mg/ml pronase and 10 mg/ml hyaluronidase antigen retrieval treatment. Cytokeratin 19 (mouse monoclonal antibody, 10 μg/ml; ab9221, Abcam) IHC was performed without any antigen retrieval. Cytokeratin 8 (mouse monoclonal antibody, 10 μg/ml; MA1‐19037, Thermofisher) and PAX1 (rabbit polyclonal, 4 μg/ml; ab203065, Abcam) IHC was performed with 10 mM citrate buffer (pH 6, 70°C) antigen retrieval for 60 and 30 min, respectively. As negative control in all IHCs, normal mouse IgG₁ (X0931, Dako), or Rabbit IgG polyclonal isotype control (ab37415, Abcam) was applied at a similar concentration as the primary antibody.

Basatvat S., Bach F.C., Barcellona M.N., Binch A.L., Buckley C.T., Bueno B., Chahine N.O., Chee A., Creemers L.B., Dudli S., Fearing B., Ferguson S.J., Gansau J., Gantenbein B., Gawri R., Glaeser J.D., Grad S., Guerrero J., Haglund L., Hernandez P.A., Hoyland J.A., Huang C., Iatridis J.C., Illien‐Junger S., Jing L., Kraus P., Laagland L.T., Lang G., Leung V., Li Z., Lufkin T., van Maanen J.C., McDonnell E.E., Panebianco C.J., Presciutti S.M., Rao S., Richardson S.M., Romereim S., Schmitz T.C., Schol J., Setton L., Sheyn D., Snuggs J.W., Sun Y., Tan X., Tryfonidou M.A., Vo N., Wang D., Williams B., Williams R., Yoon S.T, & Le Maitre C.L. (2023). Harmonization and standardization of nucleus pulposus cell extraction and culture methods. JOR Spine, 6(1), e1238.

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Immunohistochemical Staining of Ki67

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Slides were dewaxed in 3 5-min xylene baths, rehydrated in 2 1-min methanol baths and rinsed for 1 min in tap water.
For haematoxylin and eosin staining, sections were then incubated with Mayer’s haematoxylin for 3 min, rinsed for 1 min in tap water, incubated with eosin for 3 min and rinsed for 1 min with tap water before proceeding to dehydration.
To stain for Ki67, slides were then rinsed in 2 2-min PBS baths, boiled for 25 min in sodium citrate buffer (5 mM citrate and 5 mM sodium citrate, pH adjusted to 6.1), rinsed for 1 min in tap water then in 2 2-min PBS baths, blocked for 10 m in 3% peroxide diluted in PBS, rinsed in 2 2-min PBS baths, blocked for 30 m in 20% rabbit serum diluted in PBS, incubated for 60 m in 1:100 anti-Ki67 antibody (Dako M7240) or 1:217 (to match concentration) IgG control (Dako X0931), rinsed in 2 2-min PBS baths, incubated for 1 h in secondary antibody (Dako P026), rinsed in 2 2-min PBS baths, incubated for 10 m in DAB (abcam ab64238), rinsed for 1 min in tap water, incubated for 3 min in haematoxylin (Sigma-Aldrich MHS16) and rinsed for 1 min before proceeding to dehydration.
Following staining, all slides were dehydrated in 3 1-min methanol baths and 2 3-min xylene baths, then coverslipped with DPX mountant.

Jones S., Ashworth J.C., Meakin M., Collier P., Probert C., Ritchie A.A., Merry C.L, & Grabowska A.M. (2023). Application of a 3D hydrogel-based model to replace use of animals for passaging patient-derived xenografts. In Vitro Models, 2(3-4), 99-111.

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Flow Cytometry Analysis of IL-17RA and IL-17RE

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Epithelial and neutrophil IL-17RA and IL-17RE expression was investigated by flow cytometry. Cells were infected as mentioned above and stained with primary mouse anti-human IL-17RA antibody (MAB177, 5 μg/ml, RnD Systems, Denmark) or rabbit anti-human IL-17RE antibody (1 μg/ml, ab77527, Abcam, see S1 Fig for optimization). The cells were washed two times in PBS and stained with secondary goat anti-mouse or anti-rabbit IgG (1:300, Alexa Flour 488, Life Technologies, Carlsbad CA, USA) antibody for 30 min on ice. Mouse IgG (5 μg/ml, X0931, Dako, Denmark) or rabbit IgG (1 μg/ml, X0936, Dako, Denmark) were used as isotype controls and unstained cells were used as negative background control. Cells were washed twice in PBS and analyzed by flow cytometry in a FACSCalibur instrument (Becton Dickinson, Oxford, UK). A total of 5000 gated events were collected per sample, and data were analysed by Cytobank software[34 (link)]. Median fluorescence intensities (MFI) were calculated and compared to uninfected control or isotype control, whichever was highest.

Tenland E., Håkansson G., Alaridah N., Lutay N., Rönnholm A., Hallgren O., Westergren-Thorsson G, & Godaly G. (2016). Innate Immune Responses after Airway Epithelial Stimulation with Mycobacterium bovis Bacille-Calmette Guérin. PLoS ONE, 11(10), e0164431.

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Quantifying Angiogenesis in Matrigel

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Twenty images from mid-Matrigel H & E sections of all animals in vehicle and propranolol groups were taken randomly at 40× (Zeiss Axiophot II, equipped with AxioCam MRc5 and supplemented with AxioVision Rel. 4.8 software). Luminal structures containing at least one RBC were counted as one vessel; the average from 20 images was expressed as vessels/mm². Formalin-fixed, paraffin-embedded mid-Matrigel sections were processed and stained with mouse anti-human CD31 (1:40, Dako M0823) or mouse IgG (1:40, Dako X0931) as described^{18 (link)}.

Lee D., Boscolo E., Durham J.T., Mulliken J.B., Herman I.M, & Bischoff J. (2014). Propranolol Targets Contractility of Infantile Hemangioma-derived Pericytes. The British journal of dermatology, 171(5), 1129-1137.

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Quantitative Analysis of Insulin and IGF-1 Receptors

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The relative expressions of IR and IGF-IR in all cell lines were determined using two methods: (i) western blotting and densitometry and (ii) a fluorescence-activated cell sorting -based receptor quantification system using QIFIKIT (indirect immunofluorescence staining in flow cytometry) (Dako, Denmark) according to manufacturer’s protocols using either the murine monoclonal antibody 83–7 against the human IR (10 ug/ml), 24–31 against the human IGF-IR (10 ug/ml), or an isotype control antibody (Dako, X0931, 10 ug/ml). Cells were then analysed using an LSRFortessa (BD Biosciences, Franklin Lakes, NJ).

Baricevic I., Jones D.R., Roberts D.L., Lutzen A., Lundby A., Worm J., Hansen B.F, & Renehan A.G. (2015). A framework for the in vitro evaluation of cancer-relevant molecular characteristics and mitogenic potency of insulin analogues. Carcinogenesis, 36(9), 1040-1050.

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Automated Immunohistochemical Staining for HIF-1α

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Methods for tissue microarray construction were previously described (Eustace et al, 2013a (link)). Immunohistochemistry was carried out for CAIX and GLUT1 as per a previous protocol (Hoskin et al, 2003 (link)). HIF-1α staining was carried out using the Bond-Max Automated staining system (Leica Biosystems, Newcastle, UK). Samples were de-waxed and rehydrated, followed by antigen retrieval at pH 9.0 for 40 min at 100 °C. Endogenous peroxidase was blocked using 3% hydrogen peroxide solution. Primary antibody (mouse monoclonal HIF-1α, BD Biosciences 610959, Oxford, UK) was diluted to a 1 : 20 solution with diluent and incubated with samples for 15 min at room temperature. A negative control of IgG1 (Dako X0931, Cambridge, UK) was also used. Post-primary rabbit anti-mouse link reagent was applied (Bond Polymer Refine Detection System, Leica DS9800, Newcastle, UK), and samples were incubated for 8 min at room temperature. The anti-rabbit polymer-HRP detection reagent (Bond Polymer Refine Detection System, Leica) was then added and samples were incubated at room temperature for a further 8 min. 3,3′-diaminobenzidine tetrahydrochloride was added, and after a further 10 min of incubation samples were counterstained with haematoxylin.

Hunter B.A., Eustace A., Irlam J.J., Valentine H.R., Denley H., Oguejiofor K.K., Swindell R., Hoskin P.J., Choudhury A, & West C.M. (2014). Expression of hypoxia-inducible factor-1α predicts benefit from hypoxia modification in invasive bladder cancer. British Journal of Cancer, 111(3), 437-443.

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Evaluating Cartilage Tissue Formation

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To macroscopically evaluate pellet size of 14-days cultured pellets, pictures were taken using a microscope (Wild M3B, Heerbrugg, Switzerland). Subsequently, pellets were fixed in 4% formalin for 14 hours, embedded in paraffin and sectioned (6 μm). Sections were stained with 0.1% aqueous Safranin O (Brunschwig Chemie, Amsterdam, the Netherlands), resulting in red-staining of negatively-charged GAGs. As counter-staining, 0.1% aqueous Fast Green (Brunschwig Chemie) was used to stain the cytoplasm blue/green.
For immunohistochemical staining of collagen type II, sections were pre-treated with 1 mg/mL pronase and 10 mg/mL hyaluronidase (Sigma-Aldrich), and then incubated with 0.4 μg/mL antibody specific for collagen type II (#II-II6B3, Developmental Studies Hybridoma Bank, Iowa City, IA, USA) or 0.4 μg/mL mouse-IgG1 (#X0931, Dako, Glostrup, Denmark). Following incubation with alkaline phosphatase (AP)-conjugated secondary antibody (1:50, #HK-321-UK, Biogenex, San Ramon, CA, USA), AP-activity was visualized (magenta color) by incubation with new-fuchsin substrate. Sections were counterstained with haematoxylin (purple) to visualize nuclei.

de Kroon L.M., Narcisi R., van den Akker G.G., Vitters E.L., Blaney Davidson E.N., van Osch G.J, & van der Kraan P.M. (2017). SMAD3 and SMAD4 have a more dominant role than SMAD2 in TGFβ-induced chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. Scientific Reports, 7, 43164.

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Immunohistochemical analysis of TGF-beta signaling

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After resection, the tissues were embedded in paraffin and 5 μm sections were cut for immunohistochemistry. Sections were processed as previously described [6 (link)], and incubated with the primary antibodies for TGFb2 (1:200, PA5–32629, Thermo Fisher Scientific Inc., Waltham, MA), TGFbR1 (abcam, ab31013, 1:200); MEK1/MAP 2 K1, Thermo Fisher Scientific, MA1–095, 1:10000) and with isotype control rabbit IgG (02–6102, Life Technologies, Carlsbad, USA) or mouse IgG, (Dako, X0931), with 1.5% normal horse serum (PK-7800, RTU Vectastain, Vector Laboratories, Burlingame, USA) in PBS. For each patient, and for each immunostaining reaction, we photographed 10 zones of epithelium selected at random, at a magnification of × 20. Analyses were performed with Image J analysis software (http://rsb.info.nih.gov/ij/). Immunostaining in epithelial cells (brown) and nucleus counterstaining (purple) signals were separated and converted into greyscale intensities with the Colour Deconvolution plugin of Image J software. Two of the authors (GL and SV) analysed the staining blind and independently of each other. The ratio of immunostaining to counterstaining intensities was calculated for each Image.

Lezmi G., Vibhushan S., Bevilaqua C., Crapart N., Cagnard N., Khen-Dunlop N., Boyle-Freyssaut C., Hadchouel A, & Delacourt C. (2020). Congenital cystic adenomatoid malformations of the lung: an epithelial transcriptomic approach. Respiratory Research, 21, 43.

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