Female humanized-BLT mice were anesthetized with Nembutal. Rhodamine-labeled NPs or control nanoparticles (without rhodamine) in thermosensitive gel (20μl) was instilled into the mouse vagina. 1.5h or 24h later, mice were sacrificed and the FRT harvested, fixed in 4% paraformaldehyde solution (SafeFix, Fisher Science) and embedded in OCT compound (Sakura). Mouse vaginal frozen sections (5μm) were stained with monoclonal antibody for hCD45 (Dako, mouse IgG1), hCD3 (Thermo Scientific, rabbit IgG), hCD4 (GenWay, rabbit IgG), hCD8 (Dako, mouse IgG1), hCD11c (Leica, mouse IgG2a), mouse IgG1 (Dako), mouse IgG2a (Dako) or rabbit Ig (Dako) after blocking with Background Sniper (Biocare Medical). The sections were then stained with either DyLight 488-conjugated donkey anti-mouse IgG or DyLight 488-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch). All sections were finally counterstained with DAPI (Sigma) and analyzed by confocal microscopy (TCS SP2, Leica).
Mouse igg2a
Manufactured by Agilent Technologies
Sourced in United Kingdom
Mouse IgG2a is an immunoglobulin class G subtype 2a antibody produced in mice. It is used as a research tool in various immunological applications.
Automatically generated - may contain errors
Lab products found in correlation
Dylight 488 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Tcs sp2, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Mouse igg1, by Thermo Fisher Scientific (1 mentions)
Background sniper, by Biocare Medical (1 mentions)
Dylight488 conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Mouse igg1, by Agilent Technologies (1 mentions)
Alexa fluor 647 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Dab chromogen, by Agilent Technologies (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 633 goat anti mouse antibodies, by Thermo Fisher Scientific (1 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Alexa fluor 488 anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using mouse igg2a
1
Tracking Nanoparticle Vaginal Uptake
2
Immunohistochemical Characterization of Tissue Fibrosis
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Tissue was fixed in ice-cold acetone containing 2 mM phenylmethylsulfonyl fluoride and 20 mM iodoacetamide overnight at −20 °C and embedded in GMA, cut and stained as previously described62 (link), two sections at least 10 μM apart were stained for each antibody. Haematoxylin and eosin (H&E) staining was used to determine tissue morphology. Primary antibodies were used against the following antigens: anti-alpha smooth muscle actin (αSMA), clone 1A4 (0.7 µg/ml;, Dako UK, Ely, United Kingdom); anti-fibroblast surface protein (FSP), clone 1B10 (4 µg/ml; Sigma-Aldrich, Dorset, UK); anti-collagen type I (550346, 15 μg/ml, Millipore, Watford, UK); anti-collagen type III, clone FH-7A (3.125 µg/ml; Sigma-Aldrich, Dorset, UK); and appropriate isotype controls mouse IgG1, and mouse IgG2a (Dako). The sections were incubated with appropriate secondary antibody polyclonal rabbit anti mouse F(ab)2 biotinylated (2.5 µg/ml, Dako), incubated with streptavidin-biotin peroxidise complex detection system (Dako, UK) for 2 h, treated with chromogen aminoethylcarbazole (AEC) giving a red reaction product, and counterstained with Mayer’s haematoxylin.
3
Flow Cytometric Analysis of HLA-A68 and TAPBPR
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HeLa and HeLa-expressing TAPBPR variants were detached from flasks using trypsin. To allow membrane recovery post-trypsination the cells were resuspended in RPMI supplemented with 10% fetal calf serum for 30 min at 37°, followed by incubation at 4°. Cells were stained with an anti-HLA-A68 specific mAb (One Lambda, Canoga Park, CA) or with the TAPBPR-specific mAb PeTe4 for 30 min at 4°. Cells were stained with a mouse IgG2a as a negative control (Dako). Following extensive washing in PBS, cells were stained with AlexaFluor 647 goat anti-mouse IgG (Life Technologies, Paisley, UK) for 30 min at 4°. Cells were analysed on a BD Bioscience FACS Calibur 4-colour analyser.
4
Immunohistochemical Detection of OXTR
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Sections were de-paraffinised and rehydrated through a series of xylene and graded ethanol solutions. Sections were placed in 1.2% H2O2 for 15 min to block endogenous peroxidase activity. For immunohistochemistry, tissue was incubated with the primary antibody OXTR (1 μg/mL; Abcam Cat# ab87312, RRID:AB_10674457), diluted in PBS with 0.2% BSA and 0.1% sodium azide for 24 h at 4 °C. Sections were washed with PBS, and incubated with an anti-goat secondary antibody (1.25 μg/mL), the Vectastain ABC-HRP Kit and DAB + chromogen (DAKO), followed by counterstaining with haematoxylin. For immunofluorescence, sections were incubated with both α-Smooth Muscle Actin (α-SMA) (5.3 μg/mL, Sigma-Aldrich Cat# A2547, RRID:AB_476701) and OXTR, and visualised using a donkey anti mouse IgG Alexa Fluor 488 secondary antibody (1:500, Molecular Probes Cat# A-21202, RRID:AB_141607), and a rabbit anti goat IgG Alexa Fluor 555 secondary antibody (1:500, Thermo Fisher Scientific Cat# A-21428, RRID:AB_2535849). Sections were incubated with DAPI (1:1000, Thermo Fisher Scientific Cat# D3571, RRID:AB_2307445). Negative control staining was performed by incubating sections with Goat IgG (Zymed Laboratories 02-6202) and/or Mouse IgG2a (Dako X094301-2).
5
HCMV Infection of Breast Cancer Cells
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MCF-7 and MDA-MB-231 cells non-infected or infected with HCMV MOI of 5 were fixed at 6 days post infection (dpi). Cells were treated in Fix &Perm Medium A and B according to the supplier`s instruction (Molecular Probes) and co-stained with primary antibodies HCMV IE (Merck) and COX-2 (CellSignaling) or 5-LO (Abcam) diluted in phosphate-buffered saline (PBS) containing 1% BSA (Sigma) for 45 min at 4 °C. After washing with PBS, cells were incubated with secondary Alexa Fluor 633 goat anti-mouse antibodies (Life Technologies) and Alexa Fluor 488 anti-rabbit antibodies (Invitrogen). Rabbit IgG (Biocare Medical) and mouse IgG2a (Dako) served as negative controls. Acquisition was performed using Flow cytometry (FACS) and data were analyzed by Summit 4.3 software. (Three separate experiments were performed).
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