PLVAP/PV-1 Antibody [E23J22]

Catalog No.: F2799

Application: Reactivity: Mouse

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代表番号: 045-509-1970|電子メール：sales@selleck.co.jp

キーポイント

この抗体には抗ラット二次抗体が必要です。

使用情報

Dilution
IHC1:1000 IF1:1000 FCM1:10000

Application
IHC, IF, FCM

Source
Rat Monoclonal Antibody

Reactivity
Mouse

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

ポジティブコントロール	Mouse brain tissue; Mouse large intestine; bEND.3 cells
ネガティブコントロール

プロトコール

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

生物学的記述

Specificity
PLVAP/PV-1 Antibody [E23J22] detects endogenous levels of total PLVAP/PV-1 protein.

タンパク質の局在
細胞膜、細胞質、細胞内膜系

Uniprot ID
Q91VC4

Clone
E23J22

Synonym(s)
Pv1; Plasmalemma vesicle-associated protein; MECA-32 antigen; Plasmalemma vesicle protein 1; PV-1

Background
PLVAP (PV-1, plasmalemma vesicle-associated protein) is a type II transmembrane glycoprotein selectively expressed in fenestrated endothelia of kidney glomeruli, intestinal villi, liver sinusoids, and tumor neovasculature, but absent from the blood-brain barrier and continuous vessels, that organizes stomatal and fenestral diaphragms via its ~390-residue C-terminal extracellular coiled-coil domain. This domain forms rigid, parallel α-helical homodimers stabilized by five interchain disulfide bonds (Cys142, Cys153, Cys178, Cys199, Cys224), radiating as 8–10 spoke-like fibers spanning caveolar necks or fenestrae with precise 10–15 nm geometry. Functioning as a molecular sieve, PLVAP selectively restricts plasma protein transcytosis through caveolae and transendothelial channels, establishing a cutoff that allows passage of albumin and IgG while blocking larger complexes. VEGF/PI3K/p38MAPK signaling transcriptionally induces PLVAP during angiogenesis to widen caveolar openings and increase diaphragm density, which paradoxically enhances paracellular leakage via endothelial contractility. Genetic ablation of PLVAP results in 2–3-fold enlarged caveolae, loss of diaphragms, protein-losing enteropathy, hypoproteinemia, hypertriglyceridemia, and neonatal lethality from circulatory collapse, underscoring its essential role in maintaining basal permeability restriction. PLVAP is upregulated by hypoxia and VEGF, marking leaky tumor microvasculature that facilitates metastasis, and mediates vasogenic edema and leukocyte diapedesis in ischemia and sepsis.

Background

PLVAP (PV-1, plasmalemma vesicle-associated protein) is a type II transmembrane glycoprotein selectively expressed in fenestrated endothelia of kidney glomeruli, intestinal villi, liver sinusoids, and tumor neovasculature, but absent from the blood-brain barrier and continuous vessels, that organizes stomatal and fenestral diaphragms via its ~390-residue C-terminal extracellular coiled-coil domain. This domain forms rigid, parallel α-helical homodimers stabilized by five interchain disulfide bonds (Cys142, Cys153, Cys178, Cys199, Cys224), radiating as 8–10 spoke-like fibers spanning caveolar necks or fenestrae with precise 10–15 nm geometry. Functioning as a molecular sieve, PLVAP selectively restricts plasma protein transcytosis through caveolae and transendothelial channels, establishing a cutoff that allows passage of albumin and IgG while blocking larger complexes. VEGF/PI3K/p38MAPK signaling transcriptionally induces PLVAP during angiogenesis to widen caveolar openings and increase diaphragm density, which paradoxically enhances paracellular leakage via endothelial contractility. Genetic ablation of PLVAP results in 2–3-fold enlarged caveolae, loss of diaphragms, protein-losing enteropathy, hypoproteinemia, hypertriglyceridemia, and neonatal lethality from circulatory collapse, underscoring its essential role in maintaining basal permeability restriction. PLVAP is upregulated by hypoxia and VEGF, marking leaky tumor microvasculature that facilitates metastasis, and mediates vasogenic edema and leukocyte diapedesis in ischemia and sepsis.

References
[1] Chang TH, et al. Proc Natl Acad Sci U S A. 2023 Apr 4;120(14):e2221103120. [2] Stan RV, et al. Mol Biol Cell. 2004 Aug;15(8):3615-30.

PVDFメンブレン孔径参考表

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%