Immunofluorescent Detection of Mucin 2

To detect mucin 2 (MUC2), Carnoy-fixed samples were cut into 5-µm-thick sections, mounted on adhesive microscope slides (SuperFrost Ultra Plus, Thermo Scientific), and rehydrated and rinsed in accordance with standard protocols^{17 (link),77 (link)}. The samples were confined (Dako Pen, Agilent Technologies) and incubated sequentially with a protein block (Dako, Agilent Technologies), a primary antibody (2 μg/mL of MUC2 rabbit polyclonal IgG, Santa Cruz Biotechnologies), and a secondary antibody (2 ng/mL of Alexafluor 568 goat red anti-rabbit IgG, Invitrogen, Thermo Fischer Scientific); both of the antibodies were diluted (Dako Diluent, Agilent Technologies). Sections were then treated with trihydrochloride trihydrate (0.5 mg/mL Hoechst 33342, Invitrogen, Thermo Fischer Scientific) in PBS. The slides were mounted using fluorescent mounting medium (Dako, Agilent Technologies). Tissues were visualized using a high-capacity digital slide scanner (3DHISTECH Ltd.) and Pannoramic Viewer and CaseViewer software (3DHISTECH Ltd.)

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Martín R., Chamignon C., Mhedbi-Hajri N., Chain F., Derrien M., Escribano-Vázquez U., Garault P., Cotillard A., Pham H.P., Chervaux C., Bermúdez-Humarán L.G., Smokvina T, & Langella P. (2019). The potential probiotic Lactobacillus rhamnosus CNCM I-3690 strain protects the intestinal barrier by stimulating both mucus production and cytoprotective response. Scientific Reports, 9, 5398.

Publication 2019

SuperFrost Ultra Plus Dako Pen Alexafluor 568 goat red anti-rabbit IgG Dako Diluent Hoechst 33342 high-capacity digital slide scanner

A protein Anti igg Antibodies Antibody Goat High Hoechst 33342 Microscope Mucin 2 Rabbit Tissues

Corresponding Organization :

Other organizations : Microbiologie de l’alimentation au service de la santé, Université Paris-Saclay, Nutricia Research (Netherlands), Iltoo Pharma (France)

Top 5 similar protocols

Variable analysis

independent variables

Carnoy-fixed samples
Protein block treatment
Primary antibody treatment (MUC2 rabbit polyclonal IgG)
Secondary antibody treatment (Alexafluor 568 goat red anti-rabbit IgG)

dependent variables

Detection of mucin 2 (MUC2)

control variables

5-µm-thick sections
Mounted on adhesive microscope slides (SuperFrost Ultra Plus, Thermo Scientific)
Rehydration and rinsing according to standard protocols
Confined samples (Dako Pen, Agilent Technologies)
Antibody dilutions (Dako Diluent, Agilent Technologies)
Hoechst 33342 (0.5 mg/mL) treatment in PBS
Fluorescent mounting medium (Dako, Agilent Technologies)
Tissue visualization using a high-capacity digital slide scanner (3DHISTECH Ltd.) and Pannoramic Viewer and CaseViewer software (3DHISTECH Ltd.)

positive control

Not explicitly mentioned.

negative control

Not explicitly mentioned.

Annotations

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