Cat. # | Size | Qty. | Price | Inventory |
---|---|---|---|---|
54505S | 100 µl |
|
REACTIVITY | H |
SENSITIVITY | Endogenous |
MW (kDa) | |
Source/Isotype | Mouse IgG2b |
Product Information
Application | Dilution |
---|---|
IHC Leica Bond | 1:400 - 1:1600 |
Immunohistochemistry (Paraffin) | 1:100 - 1:400 |
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 | Polymer 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. | |
6 | Post Primary Mouse Linker | Polymer Refine Detection Kit Post Primary* | 10 min. |
WASH | BOND™ Wash Solution | 3x 2:00 min. | |
7 | Secondary Detection | Polymer Refine Detection Kit Polymer* | 10 min. |
WASH | BOND™ Wash Solution/Deionized Water | Custom (see below) | |
8a | Visualization | Polymer Refine Detection Kit Mixed DAB Refine* | 0:00 min. |
8b | Visualization | Polymer Refine Detection Kit Mixed DAB Refine* | 10 min. |
WASH | Deionized Water | 3x 0:00 min. | |
9 | Counterstain | Polymer 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. | |
10 | 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. | |||
11 | Mount sections with coverslips and #14177 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: 1445
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, Mouse) #8125 | SignalStain® Boost IHC Detection Reagent (AP, Mouse) #31926 |
---|---|---|
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: 280
Human
Monoclonal antibody is produced by immunizing animals with a prokaryotic recombinant protein corresponding to a region of the N-terminal intracellular domain of human CD71 protein.
Transferrin receptor 1 (CD71, TFRC) is a type II transmembrane receptor and carrier protein responsible for the uptake of cellular iron through receptor-mediated endocytosis (1). Neutral pH at the cell surface promotes binding of the iron-binding glycoprotein transferrin (Tf) to the CD71 receptor. The receptor-ligand complex enters the cell through receptor-mediated endocytosis and is internalized into an endosome. Relatively lower endosomal pH leads to a change in the local charge environment surrounding the iron-transferrin binding site and results in the release of iron (2). The receptor-ligand complex is recycled to the cell surface where transferrin dissociates from the CD71 receptor (2). Ubiquitously expressed transferrin receptor is continuously recycled and undergoes clathrin-mediated endocytosis regardless of ligand binding state. The interaction between receptor and ligand has been studied in detail. The helical domain of CD71 directly interacts with the transferrin C-lobe and induces a conformation change in Tf to facilitate the transport process (3). Interaction between the receptor CD71 and transferrin is mediated by the membrane protein hemochromatosis (HFE). HFE binds the α-helical domain of CD71, blocking formation of the CD71-transferrin complex and inhibiting iron uptake (4,5). In addition to binding transferrin, CD71 also interacts with H-ferritin at the cell surface and transports this intracellular iron storage protein to cellular endosomes and lysosomes (6). Additional studies indicate that the transferrin receptor is an evolutionarily conserved receptor for a number of arenaviruses and at least one retrovirus (7,8). Aberrant expression of CD71 is seen in a number of cancers, including thyroid carcinomas, lymphomas, and T-lineage leukemias, suggesting a possible therapeutic role for targeted inhibition of the transferrin receptor (9,10).
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