IMMUNOFLUORESCENCE TECHNIQUE

CD31 Antibodies

Mouse anti-human CD31 antibodies

-4 clones  

CATALOG#                 CLONE#                  Workshop    Host         Form        Price

RDI-M1536clb              CLB-HEC/75                IV            mIgG1       purified     $375.00

RDI-M1670clb                  " "                               "                "             FITC        $375.00

RDI-CD31-HEC7           HEC7                          VI            mIgG2a     purified     $562.00  (blocking)

RDI-CBL473                  WM59                         IV            mIgG1       purified     $438.00

RDI-CD31abm-B3         158-2B3                        --            mIgG1       see below forms available

RDI-CBL468                   HC1/6                         V             mIgG1       purified     $375.00 (paraffin)

RDI-CBL468FT                " "                               "                "             FITC        $375.00

RDI-CBL468PE                " "                               "                "             PE            $469.00

RDI-CD31abm-1A          1A10                           --             mIgG1     TCS          $500.00 (paraffin sections )

Mouse anti- human CD31(PECAM-1)/R-PE* clone 158-2B3

cat# RDI-CD31abm-BPE                        $500.00/120T

non conjugated: RDI-CD31abm-B3        $312.00/100ug

FITC conjugated: RDI-CD31abm-BFT   $375.00/120T

Biotin conjugated: RDI-CD31abm-BBT $375.00/100ug

Package Size: 120T in 0.2ml 50mM sodium phosphate pH 7.5, 500mM Potassium Chloride, 150mM NaCL, 15% glycerol, 0.2% BSA and 0.04% NaN3.

Species: mouse IgG1

mAb name/Clone: 158-2B3

Immunogen: Stimulated human leukocytes

PRODUCTION: Protein A purified antibody from tissue culture supernatant was conjugated to R-Phycoerythrin through a sulfo-ester linkage. Unconjugated antibody was removed using size exclusion chromatography. Antibody conjugate is at 0.5mg/ml with an A565/A280 ratio of 3.19 (see batc sheet for bacth specific data)

WORKING DILUTION: 1:50 (80ul/test)

INFORMATION: Human CD31 is an adhesion molecule expressed on platelets, endothelial cells, leukocytes and their bone marrow precursors. CD31 plays a role in homophilic adhesion and heterophilic transendothelial migration (3).Antibody 158-2B3   reacts with domain 1 of CD31 and blocks homophilic interaction and heterophilic transendothelial migration (1).

Reference: 1) Leukocyte Typing VI (T. Kishimoto, et al, eds.) Garland Publishing, Inc., New York (1997) p. 362-372. 2) P.J. Newman, (1997) J Clin Invest 99: 3-10. 3) K.L. Yong, et al, (1998) Blood 91: 1196-1205.

STORAGE : Store at 2 - 5oC. Do not freeze! Protect from light.

PERFORMANCE: 500K cultured Jurkat cells were washed and incubated 45 minutes on ice with 80 ul of anti-CD31/R-PE at a 1:50 dilution (10 ug/ml). Cells were washed three times, fixed and analyzed using a BD FACS Calibur. Cells stained continued on back side positive with a mean shift of 1.87 log10 fluorescent units when compared to a Mouse IgG1/R-PE negative control (Catalog #RDI- MIGG1XPE $188.00) at a similar concentration. Binding was 100% blocked when pre incubated 10 minutes with 20 ml of 0.5 mg/ml anti-CD31 antibody (Catalog RDI-CD31abm-B3).

R-PE is covered under patents US 4,520,110; European 76,695 and Canadian 1,179.942.

It is the responsibility of the user to comply with all local/state  and Federal rules in the use of this product. We are not responsible for any patent infringements that might result with the use of or derivation of this product.

For Research Use Only

Product Specification: mouse monoclonal CD31

PeliCluster  CD31

CAT#RDI- M1536clb  $375.00/vial

Test/vial 200

Clone CLB-HEC/75   This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human endothelial cells. This antibody has been clustered to CD31 in one of the international Workshop on Human White Cell differentiation Antigens.

Isotype Mouse IgG1.

Source Ascites fluid of tumour bearing BALB/c mice.

Purification Affinity chromatography.

Packing Each vial contains 1 ml with approximately 0.2 mg/ml monoclonal antibody and 10 mg BSA in 20 mM TRIS and 150 mM NaCl, pH 8.0.

Preservative Mertiolate (0.001%).

Storage and stability Monoclonal antibodies should be stored in the dark at 2-8°C. The reagent is stable until the expiry date stated on the vial label.

Major reactivity The monoclonal antibody is directed against the CD31- antigen (gpIIa', PECAM-1), which is expressed on human thrombocytes. The monoclonal antibody reacts with thrombocytes, monocytes/macrophages, granulocytes and B-cells. In immunohistology the monoclonal antibody reacts with endothelial cells histiocytes (weak) and glomeruli.

Molecular mass 140 kDa.

Application Functional studies on cells.

Methods Indirect immunofluorescence staining with analysis by flowcytometry or fluorescence microscopy.

APPLICATION  FOR AN INDIRECT IMMUNOFLUORESCENCE TECHNIQUE

Reagents

- FITC-conjugated goat anti-mouse immunoglobulin antiserum (M1204).

   Remove aggregates by centrifugation at 1000 x g for 10 minutes.

- Buffer: Phosphate Buffered Saline (PBS), containing 0.2% Bovine Serum Albumin (BSA).

- PFA: Para-Formaldehyde 1%, pH 7.2, containing 0.2% BSA.

- microwell plates (96 wells, V bottom) or tubes.

Procedure

1 Fixate the cells with PFA 1% and wash them with buffer.

2 Prepare a mononuclear cell suspension with a concentration of 1 x 10 7 cells/ml in buffer.

3 Add 45 µl of cell suspension to the microwell wells or tubes.

4 Add 5 µl monoclonal antibody to the microwell wells or tubes and mix gently.

5 Incubate for 30 minutes at 2-8 C.

6 Add 100 µl buffer to the microwell wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

7 Aspirate the supernatant from the cell pellet and resuspend the cells.

8 Add 200 µl buffer to the microwell wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

9 Aspirate the supernatant from the cell pellet and resuspend the cells.

10 Add 50 µl FITC-conjugated goat anti-mouse immunoglobulin serum, solid phase adsorbed with human immunoglobulins,     diluted 1:80.

11 Incubate for 30 minutes at 2-8 C.

12 Repeat step 6-9.

13 Add 200 µl buffer.

14 The number of positive cells can be determined by flowcytometry analysis or by means of fluorescence microscopy.

NOTE: Care should be taken when drawing blood to avoid activation of platelets.

Platelets activation markers are weakly expressed on platelets prepared from directly fixed normal whole blood.

The expression is maximal on thrombin (1 U/ml) stimulated washed platelets.

FOR IN VITRO RESEARCH USE ONLY

Product Specification: mouse monoclonal CD31,conjugated to Fluorescein

PeliCluster  CD31 F

CAT#RDI- M1670clb

Test/vial 100

Clone CLB-HEC/75

This clone has been derived from hybridization of SP2/0 cells with spleen cells of a BALB/c mouse immunized with human endothelial cells. This antibody has been clustered to CD31 in one of the international Workshop on Human White Cell differentiation Antigens.

Isotype Mouse IgG1.

Source Ascites fluid of tumour bearing BALB/c mice.

Purification Affinity chromatography.

Conjugation Conjugated with fluorescein iso thiocyanate isomer 1 (FITC).

Molecular F/P ratio between 6.0 - 10.0.

Packing Each vial contains 1 ml FITC conjugated monoclonal antibody 10 mg BSA in PBS.

Preservative Mertiolate (0.001%).

Storage and stability Monoclonal antibodies should be stored in the dark at 2-8°C. The reagent is stable until the expiry date stated on the vial label.

Major reactivity The monoclonal antibody is directed against the CD31- antigen (gpIIa', PECAM-1), which is expressed on human thrombocytes. The monoclonal antibody reacts with thrombocytes, monocytes/macrophages, granulocytes and B-cells. In immunohistology the monoclonal antibody reacts with endothelial cells histiocytes (weak) and glomeruli.

Molecular mass 140 kDa.

Application Functional studies on cells.

Methods Direct immunofluorescence staining with analysis by flowcytometry or fluorescence microscopy.

References

1. Mourik, J.A. et al., J. Biol. Chem., 260, 11300 (1985).

2. Newman, P.J. et al., Science, 247, 1219 (1990).

APPLICATION in direct Immunofluorescence techniques

Method with ficoll purified cells

Reagents and materials

- FITC-conjugated monoclonal antibodies.

- Buffer: Phosphate Buffered Saline (PBS), containing 0.2% Bovine Serum Albumine (BSA)

- PFA: Para-Formaldehyde 1%, pH 7.2, containing 0.2% BSA

- microwell plates (96 wells, V bottom) or tybes.

Procedure

1 Prepare a mononuclear cell suspension with a concentratation of 1 x 10 7 cells/ml.

2 Ad 40 µl of cell suspension to microwell wells or tubes.

3 Add 10 µl of the antibody to the microwell wells or tubes and mix gently.

4 Incubate for 30 minutes at 2-8°C.

5 Add 150 µl buffer to the microwell wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

6 Aspirate the supernatant from the cell pellet and resuspend the cells.

7 Add 200 µl buffer to the microwell wells or 2 ml buffer to the tubes and centrifuge at 500 x g for 5 minutes.

8 Aspirate the supernatant from the cell pellet and resuspend the cells.

9 Cytoflow fluorometer analysis:

   Add 200 µl buffer to the microwell wells and transfer this final cell suspension to appropriate testtubes, or add 200 µl            buffer to the tubes.

10 If analysis within 8 hours is not possible add at no. 9, instead of buffer, 200 µl PFA 1%.

Fig. 1

Fluorescense profile of ficoll purified normal cells

(Scatter gates set on the monocyte fraction)

WHOLE blood method

Due to sophisticated laser flowcytometry we are now able to distinguish populations of human lymphocytes, monocytes and granulocytes by their characteristics in different light scattering. By gating on the appropriate cell cluster (Fig.2) we can easily determine mononuclear cells, which have been immunofluorescently stained with monoclonal antibodies, and enumerate them.

Reagents and materials

- FITC-conjugated monoclonal antibodies.

- Lysing solution (NH 4Cl, pH 7.2).

- Buffer: PBS, containing 0.2 % BSA.

- PFA: Para-Formaldehyde 1%, pH 7.2, containing 0.2 % BSA.

- Tubes.

Procedure

1 Draw blood into a blood collection tube containing EDTA.

2 Deliver 100 µl (*) of well mixed whole blood to the bottom of the testtube.

3 Add 10 µl of the undiluted antibody to the bottom of the testtube, and mix gently.

4 Incubate for 30 minutes at 2-8°C or in an ice-water bath.

5 Mix the tubes and add 2 ml of lysing solution.

6 Incubate for 3-5 minutes at room temperature.

7 When lysing is completed, centrifuge the tubes at 500 x g for 5 minutes.

8 Aspirate the supernatant from the cell pellet, resuspend the cells in 1 ml buffer.

Place the tubes in the ice-water bath.

9 Analyse the samples within 8 hours.

If analysis within 8 hours is not possible centrifuge the tubes at 500 x g. Aspirate the supernatant from the cell pellet and resuspend the cells in 1 ml PFA 1%.

Fig. 2:

Scatter patern of a whole normal blood sample

R2 - lymphocytes R3 - monocytes R4 - granulocytes

Note:Use our special negative control to determine background fluorescence produced due to Fc binding capacities by mononuclear cells.The concentration and F/P ratio of our control has been adjusted to our FITC-conjugated monoclonal antibodies.* This method was developed for blood samples with a normal white count. It may be necessary to adjust the quantity of blood for samples with very high or low white counts

FOR IN VITRO RESEARCH USE ONLY

DATA SHEET: mouse anti-human CD 31 (PECAM-1)

 cat#RDI-CD31HEC7  $562.00/vial  

Package Size: 200ug purified, sterile filtered,   preservative, carrier free in 200ul   PBS buffer

Species: mouse IgG2a

CLONE: HEC7

Activity: Reacts with human CD31 (PECAM-1)

Application: 1) Indirect immunofluorescence cell surface staining.

                     2) Blocking

                     3) immunohistochemistry

                    4) immunoprecipitation

Storage: Store at 4 DEG C. Avoid frequent freeze thaw cycles. Maximum long term stability with aliquoting and storage at -20 DEG C.

Precautions: For In vitro research Use Only. Not for use in or on humans or animals or for diagnostics.

References: -J. Immunol 1996, 156:326-335

                    -J. EXP MED ,AUGUST 1989,170:399-414

                    -Leucocyte Typing VI, Garland -Publishing, NY, 1997 pg 370-372

It is the responsibility of the user to comply with all local/state and Federal rules in the use of this product. We are not responsible for any patent infringements that might result with the use of or derivation of this product.

MOUSE ANTI-HUMAN CD31 ANTIGEN (PECAM-1)

PRODUCT CODE :RDI-CBL 473     $438.00/vial 200ug

CLONE  WM59

ISOTYPE  IgG1

IMMUNIZING ANTIGEN   Human myeloid leukaemic cells

FUSION PARTNER  NS1 myeloma cell line

SOURCE  Mouse ascitic fluid

PURIFICATION METHOD Ammonium sulphate precipitation followed by hydrophobic interaction chromatography

SPECIFICITY   :The antibody reacts with a 130-140 kDa transmembranous glycoprotein that is expressed widely amongst cells of the vascular compartment. CD31 is expressed in platelets, cultured endothelial cells, monocytes, normal tissue macrophages in tonsil, lung and kidney as well as in skin biopsies from conditions such as sarcoidosis and lepromatous leprosy.

APPLICATIONS

*This antibody reacts with the platelet gpIIa molecule (also called PECAM-1) which is also expressed on endothelial cells

*Suitable for immunoprecipitation studies

*Suitable for flow cytometry by indirect labelling

REFERENCES

Leucocyte Typing IV, Oxford University Press (1989)

Leucocyte Typing V, Oxford University Press (1995)

DeLisser, H. M. et al. Immunol. Today 15, 490-95 (1994)

Newman, P. J. et al. Science 247, 1219-22 (1990)

PRESENTATION The monoclonal is presented at a concentration of 200µg/2ml in phosphate buffered saline containing 10mM sodium azide and 1mg/ml bovine serum albumin. We recommend that each laboratory determine an optimum working titre for use in its particular application.

STORAGE For use within 1 month of purchase store at +4oC, for long term storage aliquot antibody into small volumes and store at -20oC.

For Research Use Only. Not supplied for use in diagnostic or therapeutic procedures.

Mouse ANTI-HUMAN CD31 ANTIGEN

PRODUCT CODE :RDI-CBL468   $375.00/vial 200ug

  Also available FITC labeled: cat#RDI-CBL468FT     $375.00/100 Tests

                            PE labeled: cat#RDI-CBL468PE     $469.00/100 Test

CLONE                                 HC1/6

ISOTYPE                              IgG1

IMMUNIZING ANTIGEN     PMA - treated U937 cells

FUSION PARTNER               P3-x63Ag8.653 myeloma cell line

SOURCE                                 Tissue culture supernatant

PURIFICATION METHOD   Protein A affinity chromatography

SPECIFICITY The antibody reacts with a 130-140 kDa transmembranous glycoprotein that is expressed widely amongst cells of the vascular compartment. CD31 is expressed on platelets, cultured endothelial cells, monocytes, normal tissue macrophages in tonsil, lung and kidney as well as in skin biopsies from conditions such as sarcoidosis and lepromatous leprosy.

APPLICATIONS * Studies of the role of CD31 in leucocyte migration into inflammatory sites and in the process of angiogenesis* This antibody will stain frozen tissue sections* Suitable for immunoprecipitation studies* Suitable for flow cytometry either indirectly or directly when labelled with FITC or R-PE* Suitable for use on formalin fixed paraffin embedded sections after protease pre-treatment

REFERENCES Carbanas, C. et al. Eur. J. Immunol 19, 1373-1378 (1989)Leucocyte Typing V, Oxford University Press (1995)Stockinger, H. et al. J. Immunol. 145, 3889-97 (1990)DeLisser, H. M. et al. Immunol Today 15, 490-95 (1994)

LOT SPECIFIC DATA

PRESENTATION The monoclonal is presented at a concentration of 200µg/2ml in phosphate buffered saline containing 10mM sodium azide. We recommend that each laboratory determine an optimum working titre for use in its particular application.

STORAGE For use within 1 month of purchase store at +4oC, for long term storage aliquot antibody into small volumes and store at -20oC.

-Bulk quotes on request-

DATA SHEET :Anti-human CD31 (PECAM-1) Monoclonal Antibody

(Paraffin Sections)

cat# RDI-CD31abm-1A    $500.00/vial

Specificity: Human CD31

Clone: 1A10

Ig Class: IgG1

Antigen: prokaryotic recombinnat fusion protein corresponding to a portion of the extracellular domain downstream of the signal sequence of the CD31 (PEACAM-1) molecule

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

Preparation: Lyophilized tissue culture supernatant containing 15mM sodium azide. Reconstitute with 1ml of sterile distilled water.

USE: Effective on paraffin wax embedded tissue using 0.01M citrate buffer solution combined with the high temperature antigen unmasking technique

frozen sections:Not effective

Protocol: Immunohistochemistry:Typical working dilution 1:50. High temperature antigen unmasking technique using 0.01M Citrate pH6.0. 60 Minutes incubation at 25'C. Standard ABC technique.

Positive controls: Breast carcinoma with vascular invasion

Staining pattern: Membrane

Storage and stability: 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, aliquot in non frost freze freezer, Avoid repeated freeze thaw cycles. Prepare fresh working dilutions daily.