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ANTIBODIES  

(anti-Human and others as indicated)

RDI Division of Fitzgerald Industries Intl  offers a wide line of  antibodies. Since no one antibody works best for all applications (neutralization, blotting, histochemistry, ELISA, etc), we offer many different types of antibodies to help solve this problem. Please inquire for other applications or types of antibodies not listed below.

cat# RDI-NARB2abm   $438.00 - Discontinued

Presentation: Lyophilized tissue culture supernatant containing 15 mM sodium azide. Reconstitute with 1 ml sterile distilled water . Let set at least 30 minutes, mix well.

Clone: 2D9

Hybridoma partner: Mouse myeloma (p3-NS1-Ag4-1).

Isotype: mIgG2a

Immunogen: Prokaryotic recombinant protein corresponding to the varible cytoplasmic loop domain.

Specificity: Human nicotinic acetylcholine receptor beta2

Use: Effective on paraffin wax embedded tissue using 1mM EDTA (pH 8.0) unmasking solution combined with high temperature antigen unmasking techniques.

Recommendations on use: Immunohistochemistry: Typical working dilution 1:5-1:10.

Overnight primary antibody incubation at 4'C. Standard ABC technique.

-not tested on frozen sections

-not recommended for Western blotting:

Positive controls: Hippocampus

Staining pattern: Neuronal

Storage: Store unopened lyophilized antibody at 4'C. Under these conditions, there is no significant loss in product performance up to the expiry date indicated on the vial label. The reconstituted antibody is stable for at least two months when stored at 4'C. For long term storage, it is recommended that aliquots of the antibody are frozen at -20'C (frost- free freezers are not recommended). Repeated freezing and thawing must be avoided. Prepare working dilutions on the day of use.

For Research Use Only

Application:

Neuronal nicotinic acetylcholine receptors contribute to cholinergic transmission in the central and peripheral nervous systems. They are involved in the control of several brain functions including the mechanism responsible for nicotine addiction and are affected early in neurodegenerative disordes of the central nervous system. Nicotinic acetylcholine receptors are also overexpressed during late foetal development at a stage when cortical architecture is still being defined. The known risk of maternal smoking in sudden infant death syndrome may suggest the involvement of nucitube receptors, Neuronal nicotinic acetylcholine receptors consist of different subunits, alpha 2-9 and beta 2-4,with differenct subtype arrangements corresponding to distinct pharmacological and functional properties. Nicotine binding occurs in differenct regions of the human brain. High binding has been observed in the thalamus, hippicamous, substantia nigra and lateral geniculate, while lower levels of binding have been observed in cerebral cortex and spinal cord. The  relationship of nicotinic receptor loss with the neuropathology of aging and dementia suggests that changes in receptors occur early in the disease processes and before cell loss, possibly indicating a link between nicotinic acetylcholine receptors loss and the initiation of pathology. Clone 2D9 may be of use in the study of nicotinic acetylcholine receptors beta, expression and changes of expression in Parkinson's disease, Alzheimer's disease and other neurodegenerative disorders.

References:

Court J and clementi F. Distribution of nicotinic subtypes in human brain. Alzheimer Disease and Associated Disorders 9(2): 6-14 (1995). McGehee D S and Role LW. Physiological diversity of nicotinic acetylcholine receptors expressed by bertebrate neurons. Annu. Rev. Physiol. 57: 521-546 (1995).

Stone T CNS neurotransmitters and neuromodulators: acetylcholine. CRC Press/ Chapter 5: 85-104 (1995).

Neff S. Dineley-Miller K. Char D. at al., Production of polyclonal antisera that recognized and distinguish between the extracellular domains of neuronal nicotinic acetylcholine receptors subunits. Journal of Neurochemistry. 64(1):332-339 (1995).

Rubboli f. Court J A, Sala C. et al., Distribution of nicotinic receptors in the human hippocampus and thalamus. European Journal of Neuroscience. 6: 1596-1604 (1994).

Schroder H. Zilles K. Luiten P G M, et al,: Human cortical neurons contain both nicotinic and muscarinic acetylcholine receptors: an immunocytochemical double-labeling study. Synapse: 4: 319-326 (1989).

1) Cut and mount sections on slides coated with "apes"

2) Deparrafinize sections and rehydrate to distilled water

3) Bring 1600ml 1mM EDTA* (pH 8.0) to a boil in a Prestige/Presto stainless steel pressure cooker, using a hot plate. Cover but do not lock lid.

4) Position slides into metal staining racks and lower into pressure cooker ensuring slides are well immersed in buffer Lock the lid. The small valve will rise.

5) When the pressure indicator valve (the large one) has risen after about 4 minutes, Incubate sections for 1 minute.

6) After timer rings, Remove pressure cooker from heat source and run under cold water with lid on. When the small valve sinks open lid and remove slides and place immediately into distilled water. DO NOT OPEN LID UNTIL THE SMALL VALVE Sinks or Vent Cover Lock Drops (see manual with your particular unit).

7) Wash sections PBS buffer (10mM phosphate, 0.15M NaCl pH 7.5) for 1 X 5   minutes (or 100mM TRIS, 0.15M NaCl, pH 7.5)

8) Place sections in 1.5% hydrogen peroxide/methanol for 10 minutes (or use   other appropriate peroxidase blocking procedure).

9) Wash sections in distilled water for 2 X 5 minutes, then wash sections in   PBS buffer for 2 X 5 minutes.

10) Place sections in normal serum for 20 minutes.

11) Cover sections with primary antibody for at least 60 minutes at room temp. (Optimum dilution, incubation time and temperature must be determined for each antibody and in each lab).

12) Wash in PBS buffer for 2 X 5 minutes

13) Incubate sections in secondary antibody for 30 minutes

14) Wash in PBS buffer for 2 X 5 minutes

15) Incubate slides in ABC complex for 30 minutes

16) Wash in PBS buffer for 2 X 5 minutes

17) Incubate slides in DAB or other suitable peroxidase substrate

18) Wash in distilled water for 2 X 5 minutes

19) Counterstain with haematoxylin (if required), dehydrate, coverslip, and mount

Material:

APES (3-aminopropyltriethoxysilane) cat#A3648 Sigma

To avoid sections becoming detached, sections should be mounted on "APES" covered slides, then dried at 37 DEG C overnight followed by drying at 56 DEG C for 60 minutes.

1mM EDTA (ph 8.0): Add 0.37g of EDTA (Sigma cat#E-5134) to 1 liter of distilled water. Adjust pH to 8.0 using 1.0M sodium hydroxide.

Tris: 10mmM Tris, 0.15M NaCl, pH 7.5.

PBS: 10mM Phosphate, 0.15M MaCl pH 7.5

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USA

phone (978) 371-6446 or (800) 370-2222

fax     (978) 371-2266

EMAIL:antibodies@fitzgerald-fii.com

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