Cat. # | Size | Price | Inventory |
---|---|---|---|
47948T | 20 µl | ||
47948S | 100 µl |
REACTIVITY | H |
SENSITIVITY | Endogenous |
MW (kDa) | 190 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:50 |
IHC Leica Bond | 1:100 - 1:400 |
Immunohistochemistry (Paraffin) | 1:100 - 1:400 |
Immunofluorescence (Immunocytochemistry) | 1:200 - 1:400 |
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 263
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
Proceed to one of the following specific set of steps.
NOTE: When using primary antibodies produced in rabbit to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) as a secondary antibody to minimize interference produced by denatured rabbit heavy chain. For proteins with a molecular weight in the range of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured mouse light chain.
When using primary antibodies produced in mouse to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (#58802) as a secondary antibody to minimize interference produced by denatured mouse heavy chain.
posted December 2007
revised October 2021
Protocol Id: 27
NOTE: Please see product datasheet or product webpage for appropriate antibody dilution^.
Step | Reagents | Time/Temperature | |
---|---|---|---|
1 | Dewax | BOND™ Dewax Solution, 100% Alcohol, BOND™ Wash Solution | Pre-programmed Leica® BOND™ |
2 | Antigen Retrieval | BOND™ Epitope Retrieval ER2 Solution | 20 min., 100˚C | Protocol: HIER 20 min with ER2 |
3 | Peroxide Block | Refine Detection Kit Peroxide Block* | 5 min. |
WASH | BOND™ Wash Solution | 3x 0:00 min. | |
4 | Protein Block (optional) | #5425 NGS or #15019 Animal-Free Blocking Solution | 20 min. |
5 | Primary Antibody^ | Dilute in #8112 SignalStain® Antibody Diluent | 30 min. |
WASH | BOND™ Wash Solution | 3x 2:00 min. | |
NA | Post Primary Mouse Linker | Refine Detection Kit Post Primary* | Not Applied |
6 | Secondary Detection | Refine Detection Kit Polymer* | 10 min. |
WASH | BOND™ Wash Solution/Deionized Water | Custom (see below) | |
7a | Visualization | Refine Detection Kit Mixed DAB Refine* | 0:00 min. |
7b | Visualization | Refine Detection Kit Mixed DAB Refine* | 10 min. |
WASH | Deionized Water | 3x 0:00 min. | |
8 | Counterstain | Refine Detection Kit Hematoxylin* | 5 min. |
WASH | Deionized Water | 0:00 min. | |
WASH | BOND™ Wash Solution | 0:00 min. | |
WASH | Deionized Water | 0:00 min. | |
9 | Dehydration (Offline): | ||
Incubate sections in 95% ethanol two times for 10 seconds each. | |||
Repeat in 100% ethanol, incubating sections two times for 10 seconds each. | |||
Repeat in xylene, incubating sections two times for 10 seconds each. | |||
10 | Mount sections with coverslips and #84583 SignalStain® Mounting Medium | ||
Optional Custom wash: | BOND™ Wash Solution | 2:00 | |
BOND™ Wash Solution | Dispenser Type: OPEN 0:00 | ||
BOND™ Wash Solution | 2:00 | ||
BOND™ Wash Solution | Dispenser Type: OPEN 0:00 | ||
BOND™ Wash Solution | 0:00 | ||
Deionized Water | 0:00 |
*Reagent included in BOND™ Polymer Refine Detection Kit (Catalog No: DS9800)
LEICA® is a registered trademark of Leica Microsystems IR GmbH.
BOND™ is a trademark of Leica Biosystems Melbourne Pty. Ltd. No affiliation or sponsorship between CST and Leica Microsystems IR GmbH or Leica Biosystems Melbourne Pty. Ltd is implied.
posted August 2018
revised September 2018
Protocol Id: 1444
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
RECOMMENDED DETECTION REAGENTS |
SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114 | SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653 |
---|---|---|
COMPATIBLE CHROMOGEN |
SignalStain® DAB Substrate Kit #8059 | SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713 |
SignalStain® Vivid Purple Peroxidase Substrate Kit #96632 | SignalStain® Ultra Blue Alkaline Phosphatase Substrate Kit #12824 | |
SignalStain® Deep Black Peroxidase Substrate Kit #72986 | ||
SignalStain® Radiant Yellow Peroxidase Substrate Kit #69644 |
NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.
posted February 2010
revised June 2020
Protocol Id: 283
Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our #12727 Immunofluorescence Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised November 2013
Protocol Id: 24
Human
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro403 of human CD248 protein.
CD248, also known as Endosialin and TEM1, is a stromal cell marker expressed on activated mesenchymal cells including fibroblasts and pericytes. Not normally detectable in most adult tissues, it is highly expressed in lymphoid tissues during development, and in disease states where increased stromal cell proliferation and migration are evident (1-3). CD248 is known to be upregulated in breast cancer, brain tumors (4-6), and other malignancies including sarcoma (12), and it has been implicated in sprouting angiogenesis and vasculogenesis (7). It is thought that the CD248 gene is upregulated in response to hypoxic conditions in the tumor environment through HIF2 activation (8). Interestingly, CD248 is found to be highly expressed in activated fibroblasts. In liver fibrosis for example, CD248 marks the Hepatic Stellate Cells, the activated cells responsible for matrix production (9,10). In kidney fibrosis, CD248 marks the key effector cells within the fibrotic stroma including pericytes, myofibroblasts, and stromal fibroblasts (11), and in Idiopathic Pulmonary Fibrosis, CD248 may be a marker for severity of disease (12). CD248 has become a target of interest for pharmaceutical intervention in a wide scope of diseases (13).
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