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NKCC1 (D2O8R) XP® Rabbit mAb (Rodent Specific; IF Specific) #85403
Confocal immunofluorescent analysis of mouse small intestine (positive, left) or liver (negative, right), using NKCC1 (D208R) XP® Rabbit mAb (Rodent Specific; IF specific) (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).Learn more about how we get our images
Confocal immunofluorescent analysis of rat cerebellum (positive, left) and liver (negative, right) using
NKCC1 (D208R) XP® Rabbit mAb (Rodent Specific; IF specific) (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).Learn more about how we get our images
Gallery: NKCC1 (D2O8R) XP® Rabbit mAb (Rodent Specific; IF Specific) #85403
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
- Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
- Blocking Buffer: (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) to 9.5 ml 1X PBS) and mix well. While stirring, add 30 µl Triton™ X-100.
- Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
- Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412
- Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 555 Conjugate) #4413
- Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) #8889
- Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414
NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.
- Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
B. Specimen Preparation - Frozen/Cryostat Sections (IF-F)
- For fixed frozen tissue proceed with Immunostaining (Section C).
- For fresh, unfixed frozen tissue, please fix immediately, as follows:
Cover sections with 4% formaldehyde dilute in 1X PBS.
NOTE: Formaldehyde is toxic, use only in fume hood.
- Allow sections to fix for 15 minutes at room temperature.
- Aspirate liquid, rinse three times in 1X PBS for 5 minutes each.
- Proceed with Immunostaining (Section C).
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.
- Block specimen in Blocking Buffer for 60 min.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 min each.
- Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
- Rinse three times in 1X PBS for 5 min each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
- For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.
posted November 2006
revised July 2016
NKCC1 (D2O8R) XP® Rabbit mAb (Rodent specific; IF specific) recognizes endogenous levels of total NKCC1 protein.Species Reactivity: Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of mouse NKCC1 protein.
The electroneutral cation-chloride-coupled co-transporter (SLC12) gene family comprises bumetanide-sensitive Na+/K+/Cl- (NKCC), thiazide-sensitive Na+/Cl-, and K+/Cl- (KCC) co-transporters. SLC12A1/NKCC2 and SLC12A2/NKCC1 regulate cell volume and maintain cellular homeostasis in response to osmotic and oxidative stress (1). The broadly expressed NKCC1 is thought to play roles in fluid secretion (i.e. salivary gland function), salt balance (i.e. maintenance of renin and aldosterone levels), and neuronal development and signaling (2-7). During neuronal development, NKCC1 and KCC2 maintain a fine balance between chloride influx (NKCC1) and efflux (KCC2), which regulates γ-aminobutyric acid (GABA)-mediated neurotransmission (3). Increased NKCC1 expression in immature neurons maintains high intracellular chloride levels that result in inhibitory GABAergic signaling; KCC2 maintains low intracellular chloride levels and excitatory GABAergic responses in mature neurons (4,5,8). Deletion of NKCC1 impairs NGF-mediated neurite outgrowth in PC-12D cells while inhibition of NKCC1 with bumetanide inhibits re-growth of axotomized dorsal root ganglion cells (6,7). Defective chloride homeostasis in neurons is linked to seizure disorders that are ameliorated by butemanide treatment, indicating that abnormal NKCC1 and NKCC2 expression or signaling may play a role in neonatal and adult seizures (9-12). NKCC1 is found as a homodimer or within heterooligomers with other SLC12 family members. This transport protein associates with a number of oxidative- and osmotic-responsive kinases that bind, phosphorylate, and activate NKCC1 co-transporter activity (13-16). In response to decreased intracellular chloride concentrations, Ste20-related proline-alanine-rich kinase (SPAK) phosphorylates NKCC1 to increase co-transporter activity and promote chloride influx (16-19). Oxidative stress response kinase 1 (OSR1) also phosphorylates and activates NKCC1 in response to oxidative stress (14).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. DRAQ5 is a registered trademark of Biostatus Limited. DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.