rev:      June 7, 2007

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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, 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.


LYVE-1  see 2 anti-human and 2 anti-mouse  forms  and sample staining protocols

-sample facs experiments with podoplanin and slyve  facs-lec.pdf

"isolation of lymph endothelial cells (LEC) from dermal skin capillary ECs"

below  (see also anti-PROX-1  and Podoplanin)


Rabbit Anti-human LYVE-1

Description: Produced from sera of rabbits immunised with highly pure recombinant human soluble LYVE-1 produced in insect cells. The recombinant soluble LYVE-1consists of amino acid 24 (Ser) to 232 (Gly) and is fused to a C-terminal His-tag (6xHis).

LYVE-1 has been identified as a major receptor for HA (extracellular matrix glycosaminoglycan hyaluronan) on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.

Host species:      Rabbits

Antigen:               Recombinant human soluble Lyve-1

Purification:         Protein-A Chromatography (+his tag depleted)

Stabilizer:            none

Buffer:                 lyophilized from PBS, pH 7.4 w/o preservative

Formulation:      lyophilized rabbit IgG

Reconstitution: The lyophilized IgG is stable at 4°C. for at least one month and for greater than a year when kept at -20°C. When reconstituted in sterile water/PBS to a concentration of >0.5 mg/ml the antibody is stable for at least six weeks at 2-4°C. Avoid repeated freeze-thaw cycles.

Applications

ELISA: Use at 1-15 µg/ml.

Western Analysis: Use at a concentration of 1-2 µg/ml with the appropriate secondary reagents.

Immunohistochemistry: Use at 6-30 µg/ml.

Optimal dilutions should be determined by each laboratory for each application.

Usage: Anti-human Lyve-1 is offered for research use. Not for drug use. Not for human use.

Catalogue number: RDI-102PA50     Size: 200 µg    $535.00

Literature: [Carriera et al., Cancer Res 61:8079, 2001; Jackson DG Trends Cardiovasc Med 13:1, 2003; Sleeman et al., Microsc Res Tech 55:61, 2001 ; Mäkinen et al., EMBO J 20 : 4762, 2001]

** please note : always centrifuge vials before opening **

For InVitro research use only


Anti-human LYVE-1 (Antigen-Affinity Purified)

cat#RDI-102PA50AGS        Size: 50ug        $440.00

Description: Produced from sera of rabbits immunised with highly pure recombinant human soluble LYVE-1 produced in insect cells. The recombinant soluble LYVE-1consists of amino acid 24 (Ser) to 232 (Gly) and is fused to a C-terminal His-tag (6xHis).

LYVE-1 has been identified as a major receptor for HA (extracellular matrix glycosaminoglycan hyaluronan) on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.

Host species: Rabbits

Antigen: Recombinant human soluble Lyve-1

Purification: Antigen affinity purified

Stabilizer: none

Buffer: lyophilized from PBS, pH 7.4 w/o preservative

Formulation: lyophilized rabbit IgG

Reconstitution: The lyophilized IgG is stable at 4°C. for at least one month and for greater than a year when kept at -20°C. When reconstituted in sterile water/PBS to a concentration of >0.5 mg/ml the antibody is stable for at least six weeks at 2-4°C. Recommend adding 0.1-1% BSA or other carrier to dilutions and addition of 0.05% sodium azide or other suitable preservative (if suitable for your use) Avoid repeated freeze-thaw cycles.

Applications

ELISA: Use at 1-15 µg/ml.

Western Analysis: Use at a concentration of 1-2 µg/ml with the appropriate secondary reagents.

Immunohistochemistry: Use at 6-30 µg/ml.

Optimal dilutions should be determined by each laboratory for each application.

Usage: Anti-human Lyve-1 is offered for research use. Not for drug use. Not for human use.

Literature: [Carriera et al., Cancer Res 61:8079, 2001; Jackson DG Trends Cardiovasc Med 13:1, 2003; Sleeman et al., Microsc Res Tech 55:61, 2001 ; Mäkinen et al., EMBO J 20 : 4762, 2001]

** please note : always centrifuge vials before opening **

For InVitro research use only

recombinant sLyve-1 (insect cells)

cat#RDI-S01028  $250.00/5ug

cat#RDI-S01029   $531.00/25ug


Rabbit Anti-mouse LYVE-1

Cat#RDI-103PA50S    $410.00/100ug vial Protein A purified (frozen sections/WB)

cat#RDI-103PA50AG  $406.00/50ug antigen aff pure (recommeded for paraffin sections, 0.25-0.4ug/ml)

Description: Produced from sera of rabbits immunised with highly pure recombinant mouse soluble LYVE-1 produced in insect cells. The recombinant soluble LYVE-1 consists of amino acid 24 (Ala) to 228 (Gly) and is fused to a C-terminal His -tag (6xHis). LYVE-1 has been identified as a major receptor for HA (extracellular matrix glycosaminoglycan hyaluronan) on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels  themselves.

Host species: Rabbits

Antigen: Recombinant mouse soluble Lyve-1

Purification: Protein-A Chromatography (+his tag depleted) or antigen aff pure (cat#RDI-103PA50AG)

Stabilizer: none

Buffer: lyophilized from PBS, pH 7.4 w/o preservative

Formulation: lyophilized rabbit IgG

Reconstitution: The lyophilized IgG is stable at 4°C. for at least one month and for greater than a year when kept at -20°C. When reconstituted in sterile water/PBS to a concentration of >0.5 mg/ml the antibody is stable for at least six weeks at 2-4°C. Recommend adding 0.1-1% BSA or other carrier to dilutions and addition of 0.05% sodium azide or other suitable preservative (if suitable for your use) Avoid repeated freeze-thaw cycles.

Applications

ELISA: Use at 1-15 µg/ml.

Western Analysis: Use at a concentration of 1-2 µg/ml with the appropriate secondary reagents.

Immunohistochemistry: Not determined so far! Under work!

Optimal dilutions should be determined by each laboratory for each application.

Usage: Anti-mouse Lyve-1 is offered for in vitro research use. Not for use in or on humans or animals-not for use in diagnostics.

Literature: [Carriera et al., Cancer Res 61:8079, 2001; Jackson DG Trends Cardiovasc Med 13:1, 2003; Sleeman et al., Microsc Res Tech 55:61, 2001 ; Mäkinen et al., EMBO J 20 : 4762, 2001]

** please note : always centrifuge vials before opening **


mouseLYVE-1 / Formalin-fixed Paraffin-embedded sections (cat#RDI-103PA50AG)

The protocol used for testing was the following:

- thermic epitope-desmasking in Citrate buffer,

- incubation with 1. antibody for 30 min at RT,

Development:

- 2. antibody: goat x rabbit, biotin-labelled

- Streptavidin-HRP-Conjugate

- H2O2/AEC as substrat/chromogen

Background-free coloring of the murine lymphatics was achieved with an IgG concentration of about 0,25 ug/mL in the antibody dilution buffer (with carrier protein and stabilizer).


Protocol for LYVE-1 staining on Ear using Whole Mount Histology

Below is a protocol which is used to stain large pieces of tissue and visualize complete structures. You'll notice that the vasculature looks very different in this type of preparation when compared to frozen sections, as the whole mount preparation allows one to see the complete blood or lymphatic vessels. Below is the protocol we used to stain the ear samples,

1. A 5mm x 5mm piece of the outer ear flap was placed into a 5ml polypropylene tube along with 200 ul of PBS + 1% bovine serum albumin + .1% sodium azide + 5% normal mouse serum (NMS)---(PBA/NMS).

2. The tissue was blocked using Fc block (anti-mouse CD16/32 Catalog#:RDI-MCD3216 $469.00/1pkg 0.5mg) at 8ug/ml added directly to the 200ul of PBA/NMS containing the tissue, and incubated at 4 degrees C for 10 minutes.

3. To stain for blood vessels within the ear we used anti-CD31 (clone MEC 13.3, RDI-mCD31abrt-FT $500.00/vial 0.5mg), and to stain for lymphatic vessels we used anti-murine LYVE-1 (cat#RDI-103PA50AG). The anti-CD31-FITC and anti-LYVE-1 antibodies were added directly to the mixture of 200ul PBA/NMS/Fc-block at 4ug/ml and 8ug/ml respectively. (This concentration of LYVE-1 is quite high and probably could be titrated down considerably…this is something that we plan to investigate further.). Samples were incubated on a shaker at 4 degrees C for a minimum of 1 hour.

4. Samples were washed by filling up the tube with 4mls of PBA and rotated at 4 degrees for 10-20 minutes.

5. After the first wash step, the PBA was drained and mouse anti rabbit-Cy3 (cat#RDI-211165109 $250.00) was used to detect the bound LYVE-1 antibody by adding the secondary antibody at 5ug/ml to 200ul of PBA/NMS and incubating as in step 3.

6. The samples were washed as described in step 4.

7. The tissue was mounted by placing it on a glass slide using forceps and 100ul of PBA was added to the tissue. A cover slip was then placed over the tissue and gently pressed down. Samples were visualized using 2-color fluorescent microscopy.

see   mouselyve.pdf


Double staining LYVE-1/CD31 on humanen cryo-sections

- dry cryo sections for 1h at RT

- fix sections for 10 min at -20°C in MeOH

- wash 3x3 min in PBS at RT

- block 20 min in 10% goat serum in PBS

- 1. AB over night at 4°C in 10% goat serum in PBS[anti h-LYVE-1 10µg/ml and anti h-CD31 1:50

- wash 3x3 min in PBS at RT

- 2. AB 30 min at RT in PBS

[goat anti rabbit IgG (H+L) CY3 1:500 and goat anti mouse biotin 1:500

- wash 3x3 min in PBS at RT

- staining of nucleus 10 min at RT in Hoechst Dye 1:5000 in PBS

- wash 3x3 min in PBS at RT

- cover with DAKO Fluorescent Mountingmedium


recombinant mouse sLyve-1 (insect cells)

cat#RDI-S01026  $250.00/5ug

cat#RDI-S01027  $531.00/25ug

Description: A DNA sequence encoding the extracellular domain of mouse LYVE-1 (Met1 - Gly228) was fused to a C-terminal His -tag (6xHis) and expressed in insect cells . Based on N-terminal sequence analysis, the primary structure of recombinant mature sLYVE-1 starts at Ala24. sLYVE-1 has a calculated monomeric molecular mass of about 25kDa but as a result of glycosylation, migrates at approximately 35 - 45 kDa under reducing conditions in SDS-PAGE.

LYVE-1 has been identified as a major receptor for HA (extracellular matrix glycosaminoglycan hyaluronan) on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule bindd both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.

Source: Insect cells

Molecular Weight: ~35-45 kDa

Purity: > 95%, by SDS-PAGE and visualised by silver stain

Endotoxin level: < 0.1 ng per µg of VEGF-C

Stabilizer: none

Buffer: none

Formulation: lyophilised

Reconstitution: The lyophilised sLYVE-1 is soluble in water and most aqueous buffers. The lyophilised sLYVE-1 should be reconstituted in PBS or medium to a concentration not lower than 50 µg/ml.

Stability: Lyophilised samples are stable for greater than six months at -20°C to -70°C. Reconstituted sLYVE-1 should be stored in working aliquots at -20°C. Avoid repeated freeze-thaw cycles!

Usage: sLYVE-1 is offered for research use. Not for drug use. Not for human use!

Literature: [Carriera et al., Cancer Res 61:8079, 2001; Jackson DG Trends Cardiovasc Med 13:1, 2003; Sleeman et al., Microsc Res Tech 55:61, 2001 ; Mäkinen et al., EMBO J 20 : 4762, 2001]

** please note : always centrifuge vials before opening **


For In vitro research Use Only. Not for use in or on humans or animals or for diagnostics.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 or derivation of this product.


RDI Div of Fitzgerald Industries Intl

(Research Diagnostics Inc)

34 Junction Square Drive

Concord MA 01742-3049

USA

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

fax     (978) 371-2266

email: sales@researchd.com

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