Tissue samples of PDGF‐B‐ or vector‐transfected LLC and T241 tumors were cut into small pieces and incubated with 1.5 mg/mL type I (17018029, Gibco) and 1.5 mg/mL II collagenase (17101015, Gibco) in PBS at 37°C for 40‐60 min. After filtering with a cell filter, cells were centrifugated at 1500 rpm for 10 min. Cell pellets were collected and incubated with a rabbit anti‐mouse NG2 antibody (1:50, AB5320, Millipore), a rat anti‐mouse CD31 antibody (1:50, 553370, BD Pharmingen, San Diego, CA, USA), a rat anti‐mouse PDGFRα antibody (1:50, 14‐1401‐82, eBioscience), a rat anti‐mouse PDGFRβ antibody (1:50, 14‐14012‐82, eBioscience), and a rat anti‐F4/80 antibody (1:200, NBP2‐12506, Novus Biologicals, Littleton, CO, USA), followed by species matched antibody incubation in ice, including a Cy5‐labeled goat anti‐rabbit antibody (1:200, AP132S; Millipore) or Cy5‐labeled goat anti‐rat antibody (1:200, AP183, Millipore) for 30 min. Cells were further washed and incubated with anti‐Cy5/Alexa Fluor 647‐coated microbeads (130‐091‐395, Miltenyi Biotec) for 15 min, and subsequently subjected to magnetic separation through magnetic columns. Collected positive cells were used for further study or stored in RNAlater (R0901, Sigma‐Aldrich, Burlington, MA, USA) at ‐70°C until further use.
Nbp2 12506
Manufactured by Novus Biologicals
Sourced in United States
NBP2-12506 is an anti-rabbit IgG antibody conjugated to HRP (horseradish peroxidase) for use in immunodetection applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab5320, by Merck Group (1 mentions)
Ap183, by Merck Group (1 mentions)
Ap132, by Merck Group (1 mentions)
Rnalater, by Merck Group (1 mentions)
Alexa fluor 488 labelled donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Pdgfrβ, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 labelled donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Lsm510 confocal, by Nikon (1 mentions)
C1 confocal microscope, by Nikon (1 mentions)
Ab133357, by Abcam (1 mentions)
Tyr705, by Cell Signaling Technology (1 mentions)
Ab15580, by Abcam (1 mentions)
Af1830, by R&D Systems (1 mentions)
Ab3849, by Merck Group (1 mentions)
Nis element, by Nikon (1 mentions)
Niko digital dx1200me camera, by Universal Imaging (1 mentions)
3 protocols using nbp2 12506
1
Isolation of Tumor Cell Subpopulations
2
Paraffin-fixed Tissue Immunofluorescence Staining
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The PFA-fixed tumour tissues were embedded in paraffin, cut to 5-μm-thick sections using a microtome, and transferred onto glass slides. After baking, the slides were processed through serial steps to deparaffinize in Tissue-Clear (Cat. No. 1466, Sakura) and rehydrate tissues in 99–95–70% ethanol. For cryosection staining, tissue slides were fixed with acetone (Cat. No. 32201, Sigma). Rehydrated tissues were washed in PBS, boiled in a unmasking solution and subsequently blocked with 4% serum before incubation overnight at 4 °C with primary antibodies against F4/80 (Rabbit, Cat. No. NBP2-12506, Novus Biologicals), Iba1 (rabbit, Cat. No. 019-19741, WAKO), PDGFRα (Rat, Cat. No. 14-1401-82, eBioscience), PDGFRβ (Rat, Cat. No. 14-1402-81, eBioscience), NG2 (Rabbit, Cat. No. AB5320, Millipore) or αSMA (mouse, Cat. No. M0851, DAKO). The tissue slides were incubated for 30 min with fluorescent-labelled secondary antibodies including: an Alexa Fluor 555-labelled goat anti-rat antibody (Cat. No. A21434, Invitrogen); an Alexa Fluor 488-labelled donkey anti-mouse (Cat. No. A21202, Invitrogen); and an Alexa Fluor 488-labelled donkey anti-rabbit (Cat. No. A21206, Invitrogen) antibody. The slides were thoroughly washed and mounted with Vectashield containing DAPI. The positive signals were analysed under a fluorescence microscope (Zeiss LSM510 Confocal, or Nikon C1 Confocal microscope).
3
Comprehensive Immunohistochemistry Protocol
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Formalin‐fixed, paraffin‐embedded tissues were cut in 5‐μm sections. Sections were evaluated by immunohistochemical analysis following our previously reported protocol (Sorrelle et al, 2019 ) using antibodies specific for anti‐P‐STAT3 (Tyr705, #9145, Cell Signaling, dilution 1:500), anti‐ARG1 (#93668, Cell Signaling, dilution 1:500), anti‐CD8α (#98941, Cell Signaling, dilution 1:1,000), anti‐ZAP70 (#2705, Cell Signaling, dilution 1:500), anti‐P‐SMAD2 (Ser465/467, AB3849, Millipore, dilution 1:500), anti‐IL‐6Rα (AF1830, R&D Systems, dilution 1:250), anti‐iNOS (PA1‐21054, Thermo Fisher Scientific, dilution 1:200), anti‐CD3 (PA1‐29547, Thermo Fisher Scientific, dilution 1:1,000), anti‐F4/80 (NBP2‐12506, Novus Biologicals, 1:200), anti‐FOXP3 (MAB8214, Novus Biologicals, dilution 1:500), anti‐CD11b (ab133357, Abcam, dilution 1:1,000), and anti‐Ki67 (ab15580, Abcam, dilution 1:1,000). Images were obtained with a Nikon Eclipse E600 microscope using a Niko Digital Dx1200me camera and ACT1 software (Universal Imaging Corporation). Pictures were analyzed using NIS Elements (Nikon).
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