Cat. # | Size | Price | Inventory |
---|---|---|---|
75484T | 20 µl | ||
75484S | 100 µl |
REACTIVITY | H |
SENSITIVITY | Endogenous (IHC-P), Transfected (W, F) |
MW (kDa) | 40-60 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
IHC Leica Bond | 1:100 |
Immunohistochemistry (Paraffin) | 1:100 |
Flow Cytometry (Live) | 1:800 |
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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: 10
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
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If live cell staining is desired, proceed to Section C.
NOTE: Count cells using a hemocytometer or alternative method.
posted January 2009
revised June 2020
Protocol Id: 133
Human
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro215 of human TIM-4 protein.
T cell Ig- and mucin-domain-containing molecules (TIMs) are a family of transmembrane proteins expressed by various immune cells. TIM-4 is expressed by dendritic cells and macrophages (1). Like TIM-1, TIM-4 is a receptor for phosphatidylserine (2). Interaction of TIM-4 with phosphatidylserine exposed by cells undergoing apoptosis leads to uptake of apoptotic cells (2,3). In this way, TIM-4 regulates adaptive immunity by removing apoptotic antigen-specific T cells following clearance of infection. Peritoneal macrophages from mice deficient in TIM-4 fail to effectively clear apoptotic cells leading to hyperactive T and B cells and autoimmunity (4). In addition, TIM-4 expressed by tumor-associated macrophages and dendritic cells leads to autophagy-mediated degradation of dying tumor cells, resulting in reduced antigen presentation and T cell responses (5). TIM-4 was also identified as a ligand for TIM-1, and interaction between these proteins regulates T cell proliferation (1). Finally, TIM-4 is expressed by a subset of proinflammatory B cells that produce IFN-γ and promote Th1 cell differentiation (6).
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