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Claudin-2 (E1H9O) Rabbit mAb (IHC Formulated) #28530
Immunohistochemical analysis of paraffin-embedded normal human small intestine using Claudin-2 (E1H9O) Rabbit mAb (IHC Formulated).Learn more about how we get our images
Immunohistochemical analysis of paraffin-embedded normal human kidney using Claudin-2 (E1H9O) Rabbit mAb (IHC Formulated).Learn more about how we get our images
Immunohistochemical analysis of paraffin-embedded mouse kidney using Claudin-2 (E1H9O) Rabbit mAb (IHC Formulated).Learn more about how we get our images
Gallery: Claudin-2 (E1H9O) Rabbit mAb (IHC Formulated) #28530
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- Ethanol, anhydrous denatured, histological grade (100% and 95%).
- Deionized water (dH2O).
- Hematoxylin (optional).
- Wash Buffer:
- 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
- SignalStain® Antibody Diluent (#8112).
- 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH2O.
- 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
- Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
- Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
- Substrate: SignalStain® DAB Substrate Kit (#8059).
- Hematoxylin: Hematoxylin (#14166).
- Mounting Medium: SignalStain® Mounting Medium (#14177).
NOTE: Do not allow slides to dry at any time during this procedure.
- Deparaffinize/hydrate sections:
- Incubate sections in three washes of xylene for 5 min each.
- Incubate sections in two washes of 100% ethanol for 10 min each.
- Incubate sections in two washes of 95% ethanol for 10 min each.
- Wash sections two times in dH2O for 5 min each.
C. Antigen Unmasking
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.
- Wash sections in dH2O three times for 5 min each.
- Incubate sections in 3% hydrogen peroxide for 10 min.
- Wash sections in dH2O two times for 5 min each.
- Wash sections in wash buffer for 5 min.
- Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
- Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
- Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
- Remove antibody solution and wash sections with wash buffer three times for 5 min each.
- Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
- Wash sections three times with wash buffer for 5 min each.
- Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
- Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
- Immerse slides in dH2O.
- If desired, counterstain sections with hematoxylin (#14166).
- Wash sections in dH2O two times for 5 min each.
- Dehydrate sections:
- Incubate sections in 95% ethanol two times for 10 sec each.
- Repeat in 100% ethanol, incubating sections two times for 10 sec each.
- Repeat in xylene, incubating sections two times for 10 sec each.
- Mount sections with coverslips and mounting medium (#14177).
posted February 2010
revised March 2016
Claudin-2 (E1H9O) Rabbit mAb (IHC Formulated) recognizes endogenous levels of total claudin-2 protein.Species Reactivity: Human, Mouse
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp196 of human claudin-2 protein.
Tight junctions, or zonula occludens, form a continuous barrier to fluids across the epithelium and endothelium. They function in regulation of paracellular permeability and in the maintenance of cell polarity, blocking the movement of transmembrane proteins between the apical and the basolateral cell surfaces. Tight junctions are composed of claudin and occludin proteins, which join the junctions to the cytoskeleton (1,2). The claudin family is composed of 23 integral membrane proteins, and their expression, which varies among tissue types, may determine both the strength and properties of the epithelial barrier. Alteration in claudin protein expression pattern is associated with several types of cancer (2,3). Claudin-1 is expressed primarily in keratinocytes (4) and normal mammary epithelial cells, but is absent or reduced in breast carcinomas and breast cancer cell lines (5,6).
Claudin-2 is expressed primarily in the proximal tubule of the normal mammalian kidney, where it regulates transepithelial ion (e.g., Na+, Cl-) reabsorption (7). Increased expression of claudin-2 has been reported in some cancer cell lines (8), including A549 lung adenocarcinoma cells, where its nuclear distribution was positively associated with enhanced proliferation (9).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. SignalStain is a trademark of Cell Signaling Technology, Inc.