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IHC / ICC Protocols

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Antigen Retrieval Methods

Although fixation is essential for the preservation of tissue morphology, this process can also have a negative impact on IHC/ICC detection. Fixation can alter protein biochemistry such that the epitope of interest is masked and can no longer bind to...

Chemotaxis Bioassay

Introduction R&D Systems uses chemotaxis bioassays to measure the activity of the following chemokines and neutralizing antibodies: Human Chemokines 6Ckine Fractalkine MCP-3 MPIF-1 CCL28 HCC-1 MCP-4 MPIF-2 CTACK I-309 MDC...

Chromogenic IHC Staining of Frozen Tissue Sections

View Full Protocol The following protocol has been developed and optimized by R&D Systems IHC/ICC laboratory for chromogenic IHC experiments using frozen tissue samples. This protocol provides a basic guide for the fixation, cryostat...

Chromogenic IHC Staining of Paraffin-embedded Tissue Section

View Full Protoco The following protocol has been developed and optimized by R&D Systems IHC/ICC laboratory for chromogenic IHC experiments using paraffin-embedded tissue samples. This protocol provides a basic guide for the fixation,...

Detection & Visualization of Antibody Binding

Following incubation with the primary antibody, antibody binding is visualized using an appropriate detection system. The method of detection can be direct or indirect, and may generate a fluorescent or chromogenic signal. Direct detection involves...

Direct ELISA with streptavidin-biotin detection

This method provides a general procedure for use with the majority of AbD Serotec reagents. In some ‚Äécases specific recommendations are provided on product datasheets, and these methods should always ‚Äébe used in conjunction with product and batch s...

Direct staining of intracellular antigens by Flow Cytometry

For the detection of cell cycle antigens such as Ki-67, PCNA and BrdU, methanol modification is recommended – see protocol 17 The detection of intracellular antigens requires a cell permeabilization step prior to staining. The method described bel...

Direct Staining of Intracellular Cytokines by Flow Cytometry

Note: The detection of intracellular cytokines requires a cell permeabilization step prior to staining. The method described below has been found to provide excellent results in our hands; however, other permeabilization techniques have been published,...

Fluorescence Microscopy (Direct Immunofluorescence)

For use with AbD Serotec’s directly-conjugated Fluorescein Isothiocyanate (FITC) monoclonal antibodies. PE- conjugated antibodies are not suitable as they are easily photo-bleached. This method provides a general procedure for use with the majority of ...

Fluorescent ICC Staining of Cells on Coverslips

The following protocol has been developed and optimized by R&D Systems IHC/ICC laboratory for ICC experiments using cells grown on gelatin-coated glass coverslips. This protocol provides a basic guide for the preparation, fixation, and fluorescent...

Fluorescent ICC Staining of Non-adherent Cells

The following protocol has been developed and optimized by R&D Systems IHC/ICC laboratory for fluorescent ICC experiments using cell smears. This protocol provides a basic guide for the preparation, fixation, and fluorescent staining of cell smear...

Fluorescent IHC Staining of Frozen Tissue Sections

View Full Protocol The following protocol has been developed and optimized by R&D Systems IHC/ICC laboratory for fluorescent IHC experiments using frozen tissue samples. This protocol provides a basic guide for the fixation, cryostat...

Fluorescent IHC Staining of Paraffin-embedded Tissue Sections

View Full Protocol The following protocol has been developed and optimized by R&D Systems IHC/ICC laboratory for fluorescent IHC experiments using paraffin-embedded tissue samples. This protocol provides a basic guide for the fixation,...

Gelatin-coated Slides for Histological Tissue Sections

In order for tissue sections to be retained on histological slides during staining and washing steps, slides need to be coated with adhesive compounds. Although there are a variety of such compounds, gelatin is the most frequently used for histological...

Guidance for staining with Mouse anti-Bromodeoxyuridine

AbD Serotec’s anti-BrdU Clones Bu20a and BU1/75 (ICR1) are suitable for flow cytometry applications. In some ‎cases specific recommendations are provided on product datasheets, and these methods should always ‎be used in conjunction with produc...

Heat-induced Epitope Retrieval (HIER)

Antigen retrieval can reveal epitopes masked during the preparation of tissues for staining. The protocol below describes a technique used by R&D Systems and can be used on both cryostat and paraffin-embedded sections. The protocol can be used with...

Immunoprecipitation

This protocol provides a general method suitable for the immunoprecipitation of radiolabeled antigens. Please note that the choice of radio-labeling procedure may be important in the design of an immunoprecipitation experiment. For the labeling of cell...

Indirect Immunostaining of Frozen Tissue Sections

For use with unconjugated monoclonal and polyclonal antibodies. This method provides a general procedure for use with the majority of AbD Serotec reagents. In some cases specific recommendations are provided on product datasheets, and these methods...

Making a 4% Formaldehyde Solution in PBS

The vast majority of IHC/ICC procedures employ fixation of tissues and cells using formaldehyde-based fixatives. The protocol below describes the technique for generating a 4% formaldehyde solution in PBS. The most effective fixative must be determined...

Preparation of Cells for Flow Cytometry

Reagents 1. PBS/BSA Phosphate Buffered Saline pH7.4 and 1% BSA Cells stored in liquid nitrogen Prepare PBS/BSA buffer, warm to incubation temperature. Carefully remove cells from liquid nitrogen storage. Thaw cells rapidly in a water bath (at...

Preparing Samples for IHC/ICC Experiments

Tissue and cell samples must be appropriately harvested and prepared for each IHC/ICC study. To facilitate the required incubation steps, whole tissues must be cut into ultra thin (5-10 µm) sections or cut into smaller pieces for whole mount IHC. For I...

Preventing Non-specific Staining

Once tissue or cell samples have been appropriately prepared and fixed, the samples are ready to be stained. All IHC/ICC studies are dependent on specific antibody-epitope binding, which is governed by hydrophobic interactions, ionic interactions, hydrogen...

Primary Antibody Selection & Optimization

The most important factor when designing an IHC/ICC experiment is selection of the primary antibody. In turn, the critical feature of a primary antibody is specificity for the epitope. All steps of an IHC/ICC experiment must be optimized to visualize...

Proliferation Bioassay HUVECs

Introduction R&D Systems uses HUVECs (Human Umbilical Vein Endothelial Cells) in proliferation bioassays of the following cytokines and neutralizing antibodies: Human Cytokines PD-ECGF VEGF R1 (Flt-1)/Fc chimera VEGF121 VEGF R2 (KDR)/Fc...

Proliferation Bioassay NR6R 3T3 Cells

First Published in R&D Systems 2003 Catalog Introduction R&D Systems uses NR6R-3T3 cells in proliferation bioassays to measure the activity of the following cytokines and neutralizing antibodies: Human Cytokines ß-ECGF FGF acidic ...

Proliferation Bioassay of TF-1 CellsP

Contents Introduction Materials Procedure Troubleshooting Reference Introduction TF-1 cells are a factor-dependent human erythroleukemic cell line. TF-1 cells are employed in proliferation bioassays by R&D Systems for the...

QC Protocol

R&D Systems Quality Control laboratories use these Western blotting and immunostaining protocols to show that our polyclonal and monoclonal antibodies are specific for the proteins they were raised against and to determine the sensitivity of the antibody...

Sandwich ELISA with direct detetction

This is a general procedure for use with the majority of AbD Serotec reagents recommended for indirect sandwich ELISA. In some cases specific recommendations are provided on product datasheets, and these methods ‚Äéshould always be used in conjunction wit...

Sandwich ELISA with streptavidin-biotin detection

This method provides a general procedure for use with the majority of AbD Serotec reagents. In some ‚Äécases specific recommendations are provided on product datasheets, and these methods should always ‚Äébe used in conjunction with product and batch s...

The Importance of IHC/ICC Controls

Appropriate controls are critical for the accurate interpretation of IHC/ICC results. A satisfactory IHC/ICC experimental design produces results that demonstrate that the antigen is localized to the correct specialized tissues, cell types, or subcellular...

Troubleshooting Guides

Western Blot Problem: No Bands Observed Problem: Faint Bands (Weak Signal) Problem: Extra Bands Problem: High Background Problem: Diffuse Bands Problem: White Bands (ECL method) Problem: Patchy uneven spots all over the blot ...

Troubleshooting IHC/ICC Experiments

Immunohistochemistry Technically, IHC and ICC are relatively simple and straightforward experimental methods. However, there are many variables which must be identified and optimized for each individual IHC/ICC study. Optimization of IHC/ICC may also...

Western Blot - Cell Lysate Protocol

This protocol is intended to provide a set of initial conditions for analysis of cell lysates samples by Western blot. Further optimization may be required for individual samples or analytes. Follow manufacturer's protocols for specific reagents when...

Whole Blood Protocol for Analysis of Intracellular Cytokines by Flow Cytometry

This is a rapid and simple approach to the analysis of intracellular cytokines by flow cytometry. It permits the analysis of small samples, and avoids any possibility of generating artifactual results during the separation of peripheral blood cells by...

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