PA5-32355

antibody from Invitrogen Antibodies

Targeting: CLDN5 AWAL, BEC1, CPETRL1, TMVCF

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Data presented on provider website)

Immunohistochemistry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-32355

Provider

Invitrogen Antibodies

Product name

Anti-Claudin 5 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

Heat-mediated antigen retrieval is recommended prior to staining, using a 10mM citrate buffer, pH 6.0, for 10 minutes followed by cooling at room temperature for 20 min. Following antigen retrieval, incubate samples with primary antibody for 10 min at room temperature. A suggested positive control is lung tissue.

Reactivity

Human, Mouse

Host

Rabbit

Isotype

IgG

Vial size

500 µL

Concentration

Lot Dependent

Storage

-20° C, Avoid Freeze/Thaw Cycles

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-Claudin 5 Polyclonal Antibody (Product # PA5-32355) and a 23 kDa band corresponding to CLDN5 was observed across cell lines tested and decreased upon VEGF and TGF-beta treatment. Few uncharacterized bands (*) were observed between 40-60 kDa. Whole cell extracts (30 µg lysate) of HUVEC (Lane 1), HUVEC treated with VEGF (30ng/ml for 48 Hours) (Lane 2), HUVEC treated with TGF-beta(5ng/ml for 48 Hours) (Lane 3), HeLa (lane 4), Raji (Lane 5), Mouse Heart (Lane 6), Mouse Kidney (Lane 7), Mouse Lung (Lane 8) and Mouse Placenta (Lane 9) were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005)..

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemical analysis of Claudin-5 using anti-Claudin-5 Polyclonal Antibody (Product # PA5-32355) in Lung Cancer Tissue. The recommened dilution for this antibody in immunohistochemistry applications is 1:50.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of CLDN5 was performed using 70% confluent log phase HUVEC and HeLa cells. The cells were fixed with 4% Paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 10 minutes at room temperature. The cells were labeled with Anti-Claudin 5 Polyclonal Antibody (Product # PA5-32355) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000 dilution) for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing plasma membrane localization. Panel e represents HeLa cells having no expression of CLDN 5. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.