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Authors: Kelsey Knight
Group: BioLegend
Summary: The following is a general procedure guide for preparation and staining of acetone-fixed frozen tissues using a purified, unconjugated primary antibody, biotinylated secondary antibody and streptavidin-horseradish peroxidase (SAv-HRP) and DAB detection system. Because each antigen differs in terms of requirement for fixation, amplification step, etc., it is not possible to write an inclusive protocol that will work for all antigens. The user must determine optimal conditions for each antigen of interest. Many protocols for staining individual antigens, as well as useful tips and troubleshooting guides for immunohistochemistry, can be found at the IHC World web site (https://www.ihcworld.com/).

Proper citation: Kelsey Knight 2019. Immunohistochemistry Protocol for Frozen Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.8wahxae Copy   

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Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.

Proper citation: Sam Li 2019. MojoSort™ Nanobeads Column Protocol - 1. protocols.io https://dx.doi.org/10.17504/protocols.io.69dhh26 Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: The HLA class I ELISA is an enzyme immunoassay based on the detection of β2-microglobulin subunit of HLA class I complexes, after capturing the complex through the conjugated biotin. To this end, biotinylated HLA class I complex is first captured in streptavidin coated microtiter wells. Subsequently, HRP-conjugated anti-human β2-microglobulin is added to detect intact HLA class I complexes. Only intact HLA class I complexes are recognized. Peptides with high affinity binding will be clearly detected by this ELISA technique, while peptides with a moderate to low binding affinity for HLA class I provide a moderate to non-detectable signal. This protocol is designed to evaluate the efficiency of peptide exchange when using the Flex-T™ system.

Proper citation: Kelsey Miller 2017. Flex-T™ HLA Class I ELISA Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.mcsc2we Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc. Note: This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.Scale up volumes if using 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes. Sample Preparation: Enzymatic digestion of mouse spleen is recommended to achieve the highest purity and yield of CD11c+ cells. There are several protocols published that can be applied. As a general guideline, cut mouse spleen into pieces and incubate in 0.5 mg/ml Collagenase for 30 to 60 minutes at room temperature or 37°C. Place the tube in a rocking platform with continuous agitation or gently pipette every 10 minutes. Alternatively, inject 1 ml of enzymes solution in the uncut organ. Force the tissue through a 70µm filter to prepare a single cell suspension, and wash with complete media. Resuspend cells in 0.1 mg/ml DNase 1 solution and incubate at room temperature for 10 minutes. Again, filter cells through a 70 µm filter and wash with complete media. Resuspend in complete media or MojoSort™ Buffer and keep on ice until ready to use.

Proper citation: Sam Li 2019. MojoSort™ Mouse CD11c Nanobeads Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7xjhpkn Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Immunohistochemistry Protocol for Paraffin-Embedded Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.97bh9in Copy   

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Authors: Kelsey Knight
Group: BioLegend
Summary: Reagents - Basic cell culture media: respective to the cell line used and experimental design Distilled water: Invitrogen® UltraPure™ Distilled Water (catalog number 19977015) or equivalent product from other vendor.Highthroughput (HT) Pro A or G 96 well plate or Spin Column (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)96-well collection plate: USA scientific® TempPlate non-skirted 96-well PCR plate natural (catalog number 1402-9596) or equivalent product from other vendor.QIAquick™ PCR purification kit (Qiagen®; catalog number 28104) or equivalent product from other vendor.Hypotonic Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Digestion Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Lysis Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Column Conditioning Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162  or 500163)Wash Buffer 1 (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Wash Buffer 2 (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Wash Buffer 3 (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)Elution Buffer (Chromatrap® HT ChIP-qPCR kit; catalog number 500161 or 500162 or 500163)5M NaCl: dissolve 292g of NaCl in 800mL of H2O. Adjust the volume to 1L with H2O.1M NaHCO3: dissolve 12.6g of NaHCO3 in 100 mL of H2O. Adjust the volume to 150 mL with H2O.Protease Inhibitor Cocktail (PIC): Cell signaling Technology® (catalog number 5871) or equivalent product from other vendor.Proteinase K: Qiagen® (catalog number 19133) or equivalent product from other vendor.Proteinase K stop solution: 100mM Phenylmethylsulfonyl fluoride (PMSF). Weigh 1.742g PMSF (Sigma-Aldrich®, catalog number 10837091001) and add DMSO (Sigma-Aldrich®, catalog number D2650) to a final volume of 100mL. Dissolve completely.PBS: HyClone™ Phosphate Buffered Saline (1X) (catalog number SH30256.01) or equivalent product from other vendor.Formaldehyde: VWR® 37% w/w aqueous solution (catalog number 97064-604) or equivalent product from other vendor.0.65M Glycine: Dissolve 488g of glycine into 1L of H2O to make 6.5M glycine (10X). Dilute with PBS to make 1X before each experiment.   Micrococcal Nuclease: Cell Signaling Technologies® (catalog number 10011S) or equivalent product from other vendor.1.3-1.5% Agarose gel: dissolve 1.3-1.5g of agarose powder into 100mL of TAE buffer by microwave heating. Visualize DNA under UV light by adding appropriate amount of Ethidium Bromide, or use an equivalent method.Add appropriate amount of Ethidium Bromide to cooled down gel solution to visualize DNA under UV light.0.5M EDTA: Dissolve 186.1g EDTA into 700mL of H2O. Adjust the volume to 1L with H2O.100bp ladder: NEB® (catalog number N0467S) or equivalent product from other vendor.Materials -Heat blockRotatorCentrifugeMicrocentrifuge37ºC incubatorNanodropSonicatorUV light imagerRocking platform

Proper citation: Kelsey Knight 2018. Chromatin Immunoprecipitation (ChIP) Assay Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.tj7ekrn Copy   

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Authors: Vinodkumar Kulangara, Neetha Nair, Asok Sivasailam, Suchithra Sasidharan, Justin Davis and Radhika Syam
Group: BioLegend
Summary: Dairy cows affected with subclinical mastitis can be sources of virulent, antimicrobial-resistant Staphylococci to humans by the excretion of the bacteria through their milk. This study focussed on the phenotypic and genotypic antibiotic resistance patterns of Staphylococci isolated from dairy cows during the early dry period.  Among 96 isolates of Gram positive cocci from 157 cows, 76 were identified as Coagulase Negative Staphylococci and the remaining twenty were Staphylococcus aureus. Typical amplicons of coagulase gene were obtained for all twenty samples of S. aureus with three major coagulase types being identified as giving 627 bp (40 %), 910 bp (35 %) and 710 bp (25 %) long PCR products. The groEL gene was amplified in PCR of all 76 isolates of Coagulase Negative Staphylococci, and incubation of PCR products with restriction enzyme PvuII yielded three distinct PCR-RFLP fragment patterns bearing resemblance to S. chromogenes and S. hyicus. Highest sensitivity of Coagulase Negative Staphylococci was noted for Azithromycin (92.5%) and the least to Tetracyclines (76.3%), whereas for S. aureus, it was Cefoperazone (95%) and Azithromycin (72.2%) respectively. Phenotypic resistance to Oxacillin (25 isolates), and Cefoxitin (11 isolates) was detected by dilution method with a commercial strip (Ezy MICTM).  Genotypic resistance to b-Lactam antibiotics was found in 65 (34 with mecA gene and 31 with blaZ gene) isolates. Eighteen isolates possessed both the genes, with the PVL gene for virulence being detected in five of them. Nine isolates which had mecA gene were phenotypically susceptible to oxacillin while phenotypic resistance to oxacillin was observed in seven isolates that did not have either mecA or blaZ gene. This is the first report of persistent Staphylococcal infections possessing PVL gene and high level of genotypic resistance to b-Lactam antibiotics in small- holder dairy cattle from India.  

Proper citation: Vinodkumar Kulangara, Neetha Nair, Asok Sivasailam, Suchithra Sasidharan, Justin Davis and Radhika Syam 2017. Genotypic and phenotypic ?-Lactam resistance and presence of PVL gene in Staphylococci from dry bovine udder . protocols.io https://dx.doi.org/10.17504/protocols.io.jhccj2w Copy   

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Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads than with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.

Proper citation: Sam Li 2019. MojoSort™ Human CD4 T Cell Selection Kit Column Protocol - CD4 Nanobeads. protocols.io https://dx.doi.org/10.17504/protocols.io.7tyhnpw Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. True-Nuclear™ Transcription Factor Staining Protocol for 96-Well, U-Bottom Plate. protocols.io https://dx.doi.org/10.17504/protocols.io.bacriav6 Copy   

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Authors: Kelsey Knight
Group: BioLegend

Proper citation: Kelsey Knight 2018. Th17 Polarization of Mouse CD4+ Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.tnhemb6 Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Intracellular Staining With True-Phos™ Perm Buffer in Whole Blood. protocols.io https://dx.doi.org/10.17504/protocols.io.bac6iaze Copy   

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Authors: Kelsey Knight
Group: BioLegend

Proper citation: Kelsey Knight 2018. Immunohistochemistry Protocol for Paraffin-Embedded Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.tkjekun Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: Protocols for Anti-BrdU Staining Using DNAse with Surface and Fluorescent Proteinsand Anti-BrdU Staining Using 70% Ethanol and 2N HCl

Proper citation: Kelsey Miller 2016. Anti-BrdU Staining Protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.e23bggn Copy   

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Authors: Kelsey Miller
Group: BioLegend

Proper citation: Kelsey Miller 2016. Immunohistochemistry Protocol for Ultra Streptavidin Detection Kits (USA). protocols.io https://dx.doi.org/10.17504/protocols.io.e2kbgcw Copy   

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Authors: BioLegend, Inc.
Group: BioLegend

Proper citation: BioLegend, Inc. 2016. True-Nuclear™ Transcription Factor Staining Protocol for 96-Well, U-Bottom Plate. protocols.io https://dx.doi.org/10.17504/protocols.io.exsbfne Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: This procedure is optimized for the isolation of 106 cells per test. If working with fewer than 106 cells, keep volumes as indicated for 106 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.Product description and procedure summary: Target cells are positively selected or depleted by incubating the sample with the biotin anti‐mouse CX3CR1 antibody followed by incubation with magnetic Streptavidin Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. These are the CX3CR1+ cells, do not discard them if those are the cells of interest. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.

Proper citation: Kelsey Miller 2016. MojoSort™ Mouse CX3CR1 Selection Kit Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.fyqbpvw Copy   

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Authors: BioLegend, Inc.
Group: BioLegend

Proper citation: BioLegend, Inc. 2017. Immunofluorescent Staining of Whole Blood. protocols.io https://dx.doi.org/10.17504/protocols.io.hwdb7a6 Copy   

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Authors: Kelsey Knight
Group: BioLegend

Proper citation: Kelsey Knight 2018. Preparation of Brain Samples for LEGEND MAX™ Beta Amyloid ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.tm3ek8n Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Immunohistochemistry Protocol for Beta Amyloid Products using USA Detection Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.8x4hxqw Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: Target cells are depleted by incubating your sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads (Cat. No. 480015/480016). The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.Note: This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water. Scale up volumes if using 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.Sample Preparation: Dead cell removal prior to cell isolation is recommended to achieve the highest purity and yield of mouse NK cells. Dead cells can be removed using appropriate centrifugation media or by other methods.

Proper citation: Sam Li 2019. MojoSort™ Mouse NK Cell Isolation Kit Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7yshpwe Copy   

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