Immunofluorescence Imaging of Claudin-3

Snap frozen tissue sections were fixed in 3% paraformaldehyde/2% sucrose in PBS, permeabilized with 0.5% Triton-X100, and saturated with goat serum in PBS. Samples were incubated with anti-claudin3 (Invitrogen) or with IgGH6 anti-claudin3 [13 (link)] over-night at 4°C followed by Alexa Fluor 594 anti-rabbit IgG (Molecular Probes, Eugene, OR). Cells membranes were stained with WGA lectin-Alexa Fluor 488 (Molecular Probes, Eugene, OR) and nuclei were counterstained with 4’,6-diamidino,2-phenylindole (DAPI, Sigma). Samples were analyzed using Zeiss Axiovert 200M epifluorescence microscope equipped with Apotome system, Plan-Neofluar 20x/0.5 NA and Plan-Apochromat 63x/1.4 NA oil objectives. Z-stack images were elaborated with AxioVision Inside4D module (Carl Zeiss) [24 (link)].

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Corsini M., Ravaggi A., Odicino F., Santin A.D., Ravelli C., Presta M., Romani C, & Mitola S. (2018). Claudin3 is localized outside the tight junctions in human carcinomas. Oncotarget, 9(26), 18446-18453.

Publication 2018

anti-claudin3 Alexa Fluor 594 anti-rabbit IgG 4’,6-diamidino,2-phenylindole (DAPI, Axiovert 200M epifluorescence microscope AxioVision Inside4D module

2 phenylindole Alexa 594 Alexa fluor 488 Anti igg Cells membranes Dapi Frozen sections Goat Lectin Microscope Molecular probes Nuclei Paraformaldehyde Rabbit Serum Sucrose Tissue Triton x100

Corresponding Organization :

Other organizations : University of Brescia, Yale University

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Variable analysis

independent variables

Anti-claudin3 (Invitrogen) or IgGH6 anti-claudin3 [13]

dependent variables

Claudin3 expression

control variables

Snap frozen tissue sections
Fixation in 3% paraformaldehyde/2% sucrose in PBS
Permeabilization with 0.5% Triton-X100
Saturation with goat serum in PBS
Incubation at 4°C over-night
Alexa Fluor 594 anti-rabbit IgG (Molecular Probes, Eugene, OR)
Cell membrane staining with WGA lectin-Alexa Fluor 488 (Molecular Probes, Eugene, OR)
Nuclei counterstaining with 4',6-diamidino,2-phenylindole (DAPI, Sigma)
Zeiss Axiovert 200M epifluorescence microscope equipped with Apotome system, Plan-Neofluar 20x/0.5 NA and Plan-Apochromat 63x/1.4 NA oil objectives
Z-stack images elaborated with AxioVision Inside4D module (Carl Zeiss)

controls

Positive control: Anti-claudin3 (Invitrogen)
Negative control: IgGH6 anti-claudin3 [13]

Annotations

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