Mouse anti-Amyloid Precursor Protein (monoclonal antibody)

Specificity: Human amyloid precursor protein.

Clone: 3G12

Ig Class: IgM

Immunogen: Prokaryotic recombinant fusion protein corresponding to the extracellular portion of APP between the Kunitz protease inhibitor domain and the beta amyloid region.

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

Preparation: Lyophilized tissue culture supernatant containing 15mM sodium azide. Reconstitute with 1ml of sterile distilled water . Allow to set at least 30 minutes. Mix well.

Use: Effective on pariffin wax embedded tissue: Yes (using 1mM EDTA (pH8.0) unmasking solution combined with the high temperature antigen unmasking technique for 5 minutes:

-antibody dilution 1:25-1:50.

60 minutes primary antibody incubation at 25'C.

Standard ABC technique

-not tested on frozen section

Western blotting: (dependent on the preparation of the antigen). May require reducing or non-reducing conditions. May require reducing or non-reducing conditions. (see Neil et all, 1994)

Note: We recommend the use of an anti-IgM specific conjugated secondary antibody for best sensitivity.

Pos ctrl: Alzheimer's disease brain tissue.

Stainig: Neurofibrillary tangles and senil plaques.

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 IN VITRO RESEARCH USE ONLY

Application:

Alzheimer's disease, the most common cause of dementia in the elderly, exists in both familial and sporadic forms. Genetic studies have identified three genes: beta-amyloid precursor protein (APP), Presinilin-1 and Presinilin-2 which, when mutated, can cause familial forms of Alzheimer's disease. APP and APP-like proteins are transmembrane glycoproteins with a similar modular domain structure. Clone 3G12 has been raised to the extracellular portion of APP between the Kunitz protease inhibitor domain and the beta amyloid region. This region shows the least homology with the APP-like proteins. Advantages clone 3G12 over existing antibodies to APP is that no crossreaction with APP-like proteins is evident. It reacts with late -stage neurofibrillary tangle-bearing neurons, neuritic processes surrounding senile plaques and neuropil threads in grey matter of Alzheimer's disease brain.

References:

-Neil D et al, Journal of Neuroscience Research 39:482-493 (1994)

-Ashall F and Goate A M. Role of the B-amyloid precursor protein in Alzheimer's disease. TIBS. 19: 42-46 (1994).

-Smith R P, Higuchi D A and Broze G J Jr. Platelet coagulation factor XI-inhibitor, a form of Alzheimers amyloid precursor protein. Science, 248: 1126-1128 (1990).

-Ottersdorf T, Fritz I C, Schenk D B, et al,. The secreted form of the Alzheimer's amyloid precursor protein with the Kunitz domain is protease nexin-II. Nature 341: 144-147 (1989).

-Kang J, Lemaire H-G, Unterbeck A, et al,. The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature,325: 733-736 (1987).

(suitable for western blots and for paraffin section ,using Citrate buffer release methods)

-reacts with large pyramidial cells as wells as smaller neurons, astrocytes and microglia

Sample High Temperature Unmasking Technqiue for Paraffin Sections: Buffer=1mM EDTA (ph 8.0)

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 5minutes

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