IHC was performed as described previously (11 (link)). In brief, formalin-fixed paraffin-embedded blocks of tissue were deparaffinized, rehydrated, and treated with 3% hydrogen peroxide. Antigen was retrieved, and samples were blocked in 1.5% normal horse serum. A mouse monoclonal antibody against viral protein (VP) 1 from WUPyV (WU-VP1) (NN-Ab06) or an isotype-matched control antibody (mouse IgG2b; BD Biosciences, San Jose, CA, USA; no. 557351) was incubated overnight. After incubation with the biotinylated antimouse IgG secondary antibody (Vector BA-2000; Vector Laboratories, Inc., Burlingame, CA, USA), slides were developed by using the Vectastain ABC kit (Vector Laboratories, Inc.; no. PK-6100) and DAB (Vector Laboratories, Inc.; no. SK-4100), counterstained with hematoxylin, dehydrated, cleared, and mounted.
Mouse igg2b
Manufactured by BD
Sourced in United States
Mouse IgG2b is an antibody isotype produced by mouse B cells. It binds to antigen to facilitate immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Mouse igg2a, by BD (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Lsrii system, by BD (1 mentions)
Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions)
Anti cd25 pc61.5, by BioLegend (1 mentions)
Sytox blue, by Thermo Fisher Scientific (1 mentions)
Diva software, by BD (1 mentions)
Enoxaparin, by Sanofi (1 mentions)
Mouse igg2a, by Abcam (1 mentions)
Dalteparin, by Pfizer (1 mentions)
Fitc conjugated goat anti mouse igm, by Thermo Fisher Scientific (1 mentions)
Cd1a apc mouse igg1, by BD (1 mentions)
Hla dr fitc clone g46 6, by BD (1 mentions)
Heparan sulfate clone f58 10e4, by AMS Biotechnology (1 mentions)
Af488 conjugated donkey anti mouse igg2b, by Thermo Fisher Scientific (1 mentions)
Pe mouse igg1, by BD (1 mentions)
Cold stain buffer, by BD (1 mentions)
Liquid permanent red substrate chromogen, by Agilent Technologies (1 mentions)
Aquatex, by Merck Group (1 mentions)
12 protocols using mouse igg2b
1
Immunohistochemical Detection of Viral Protein
2
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used: anti-CD5 (53–7.3), anti-CD4 (RM4-5), anti-CD8α (53–6.7), anti-TCRβ (H57-597), anti-CD44 (IM7), anti-CD3 (145-2C11), anti-CD24 (M1/69), anti-Vα2 (B20-1) all from BD Biosciences and anti-CD25 (PC61.5) from BioLegend. Cell viability was evaluated using SYTOX Blue (Life Technologies). For intracellular staining of LCK, cells were permeabilized using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit according to manufacturer protocol. Anti-LCK antibody (clone 3A5; Santa Cruz) and an isotype control antibody (mouse IgG2b from BD Biosciences) were used at 10 μg/ml. A FITC-conjugated goat anti-mouse IgG2b secondary antibody (Southern Biotechnology) was used to reveal the primary antibody. Stained cells were analysed using an LSRII system (BD Biosciences). Data were analysed with Diva software (BD Biosciences), and overlayed plots were constructed with FlowJo software.
3
Multiparametric Flow Cytometry for Glycosaminoglycans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used (all anti-human): Heparan Sulfate (clone F58-10E4) (Amsbio), digested Heparan (clone F69-3G10) (Amsbio), Syndecan 1 (DL-101) (Santa Cruz), Syndecan 2-FITC (H-7) (Santa Cruz), Syndecan 3 (M-300) (Santa Cruz), Syndecan 4 (clone F94-8G3), CD207-PE (langerin) mouse IgG1 (#IM3577) (BeckmanCoulter, USA), CD1a-APC mouse IgG1 (BD Biosciences, San Jose, CA, USA) CD1a-PE (clone SK9) mouse IgG2b (BD Bioscience), HLA-B27-FITC (clone HLA-ABC-m3), mouse IgG2a (Abcam), HLA-DR-FITC (clone G46-6), mouse IgG2b (BD Bioscience), CD80-PE, mouse IgG1 (BD Pharmingen), CD83-PE, mouse IgG1 (eBioscience), CD86-FITC, mouse IgG1 (BD Pharmingen), FITC-conjugated goat-anti-mouse IgM (#31992) (Invitrogen), AF488-conjugated goat-anti-mouse IgG1 (#A21121) (Invitrogen), AF488-conjugated donkey-anti-mouse IgG2b (Invitrogen).
The following reagents were used: Unfractionated (UF) heparin, 5.000 I.E./ml (LEO). Low Molecular Weight (LMW) heparins: dalteparin, 10.000 IE anti-Xa/ml (Pfizer), tinzaparin, 10.000 IE anti-X1/0.5ml (LEO), enoxaparin, 100 mg/ml (Sanofi). 4-Nitrophenyl β-D-xylopyranoside (PNP-Xyl, 2001-96-9) (SigmaAldrich). Heparinase III from Flavobacterium heparium, EC 4.2.2.8, Batch 010 (Amsbio). 123Count eBeads, REF# 01-1234-42, LOT# E133305, 1.011.000 eBeads/ml (eBioscience).
The following reagents were used: Unfractionated (UF) heparin, 5.000 I.E./ml (LEO). Low Molecular Weight (LMW) heparins: dalteparin, 10.000 IE anti-Xa/ml (Pfizer), tinzaparin, 10.000 IE anti-X1/0.5ml (LEO), enoxaparin, 100 mg/ml (Sanofi). 4-Nitrophenyl β-D-xylopyranoside (PNP-Xyl, 2001-96-9) (SigmaAldrich). Heparinase III from Flavobacterium heparium, EC 4.2.2.8, Batch 010 (Amsbio). 123Count eBeads, REF# 01-1234-42, LOT# E133305, 1.011.000 eBeads/ml (eBioscience).
4
Chondrocyte Surface Marker Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Monolayer cultured chondrocytes were harvested and prepared at passage 3–4 for surface marker expression by flow cytometry as previously described [25 (link)]. Briefly, chondrocytes were harvested and washed three times with cold stain buffer (Cat. no. 554656; BD Biosciences), filtered through a 70 μm cell strainer and prepared on ice as single-cell suspensions to a final concentration of < 1 × 106 cells/100 μL and incubated with antibodies at 1:10 dilution for 1 h. Fluorochrome-conjugated antibodies targeting CD44 (Cat. no. 555479), CD106 (Cat. no. 561679), CD146 (Cat. no. 561013), CD166 (Cat. no. 560903), CD271 (Cat. no. 560927), isotype control PE Mouse IgG2b (Cat. no. 555743) and isotype control PE Mouse IgG1 (Cat. no. 555749) were purchased from BD Biosciences, USA. Samples were analysed using a BD FACSAria III flow cytometer and FlowJo software (Tree Star Inc., USA). Data from three donors were presented as the average of median fluorescence intensity (MFI) +/− standard error.
5
Immunohistochemical Analysis of Small HSPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyse the expression of small HSPs, paraffin sections were double labelled for HLA-DR and small HSPs. Deparaffinised, rehydrated and endogenous peroxidase-blocked slides were heated in a microwave oven for 10 min in 0.01 M citrate buffer (pH 6.0). After antigen retrieval, sections were allowed to cool to RT and rinsed in PBS. Subsequently, sections were incubated overnight at RT with primary antibodies directed to HLA-DR and HSPB1, HSPB6, HSPB8 or HSPB11 (Table 3 ). After the sections were rinsed, the relevant secondary antibodies [goat-anti-mouse-alkaline phosphatase (AF labelled (Southern Biotech, AL, USA) or goat-anti-rabbit EnVision+™ -HRP (Dako)] were applied for 1 h at RT. Cells labelled with HRP were visualised with DAB, whereupon slides were rinsed in Tris buffered saline (TBS) to detect AF-labelled cells with liquid permanent red substrate-chromogen (Dako). The sections were counterstained with haematoxylin prior to permanent mounting in Aquatex (Merck, Darmstadt, Germany). Negative controls were performed by eliminating the primary antibodies and by replacing the primary antibodies by appropriate isotype controls, viz. mouse IgG2b (BD Bioscience, Breda, the Netherlands) or rabbit IgG (Abcam, Cambridge, UK).
6
Multicolor Flow Cytometry of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-CD19 antibody clone LT19 conjugated to APC or VioBlue was purchased from Miltenyi Biotec. Two anti–DC-SIGN antibodies and their respective isotype controls were used: anti-human DC-SIGN/CD209 clone 120507 and mouse IgG2B isotype control conjugated to APC (R&D Systems) and anti-human DC-SIGN/CD209 clone DCN46 and mouse IgG2B, κ-isotype control conjugated to V450 (BD Biosciences). The anti-EBV EA-R-p17 antibody, clone 5B11 from Millipore Sigma was used to detect expression of the early antigen encoded by BHRF1. The anti-gpK8.1A 4A4 antibody (hybridoma culture supernatant) was a kind gift from Bala Chandran, Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL.
7
PD-1 and Glut1 Expression in CD8+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sorted CD8+ T lymphocytes were resuspended in 2% phosphate-buffered saline (PBS) and stained with FITC-conjugated anti-PD-1 antibody (R&D Systems, Minneapolis, MN, USA) and FITC-conjugated anti-Glut1 antibody (Biolegend, San Diego, CA, USA). Mouse IgG2b (BD Biosciences) was used as an isotype control. The cells were incubated on ice for 30 m, washed twice with 2% PBS, and analyzed for PD-1 and Glut1 by flow cytometry.
8
Multimodal Neuroanatomical Characterization
9
Cellular Responses to Immunomodulatory Stimuli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lipopolysaccharide (LPS) (Sigma, St. Louise, MO, USA), M-CSF and GM-CSF (PeproTech, Rocky Hill, NJ, USA) were applied at final concentrations of 50 ng/ml. Recombinant human cytokines IL-4, IL-10, IL-13, IFNγ and TGF-β (R&D Systems, Minneapolis, MN, USA) were applied at final concentrations of 20 ng/ml. Antibodies against CD86, CD273, CD274, CD14, CD206 (all BD Bioscience-Pharmingen, San Diego, CA, USA), HLA-DR (eBioscience, San Diego, CA, USA) and isotype control mouse IgG2a (BD Bioscience-Pharmingen and eBioscience), mouse IgG2b (BD Bioscience-Pharmingen), were used for flow cytometric analyses. Dextran Alexa Fluor 647, 10,000 MW, anionic (Life Technologies, Stockholm, Sweden) was used for endocytosis experiments at 10 μg/ml.
10
Immunohistochemical Evaluation of CD44 and CD24 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mouse anti-human CD44 (mouse IgG2b, BD Pharmingen, CA, USA) and mouse anti-human CD24 (Thermo Scientific, MA, USA) primary antibodies were used. Immunostaining was performed using the diaminobenzidine (DAB) substrate method (Cell Signaling Technology, MA, USA) according to the manufacturer’s protocol. Before specific staining, unspecific antigenic sites were blocked with normal calf serum. Sections were then incubated with the respective primary antibody for 1 hour at room temperature (RT) followed by incubation with horse radish peroxidase single stain detection reagent (10 minutes at RT). Later, sections were washed with water for 5 minutes. Specific peroxidase activity was visualized with DAB working solution (contains DAB chromogen concentrate and DAB diluent). Counterstaining was performed with Mayers hematoxylin and eosin. A scoring scale of relative intensity of positive staining was used. A weak staining was given a score of 1, moderate staining was scored 2 and strong staining was given a score of 3. Assessment of the intensity of positive staining was performed across the entire sample in a blinded fashion upon light microscopy by two investigators and is given as the mean of both scores assessed.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!