Alexa 488 conjugated goat anti rabbit mouse igm

Either HT29-MTX cells or ileum frozen sections were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized in 0.1% Triton X-100 in PBS for 5 min, and blocked in PBS containing 5% (v/v) normal goat serum (NGS) for 30 min at room temperature. Samples were then stained with primary antibody overnight at 4 °C. Following three washes with PBS, the samples were incubated with Alexa 488-conjugated goat anti-rabbit/mouse IgM (Invitrogen Co., Carlsbad, CA, USA) and counterstained with PI in PBS containing 5% (v/v) NGS for 2 h. After washing with PBS, the samples were mounted on slides and visualized with an Olympus FluoView™ 300 confocal microscope with a 400x objective lens. For an immunohistochemical analysis, ileum frozen tissues incubated with primary antibody overnight at 4 °C were treated with a biotinylated secondary antibody solution (Vectastain Elite ABC kit, Vector Laboratories, CA, USA) for 1 h at room temperature. Sections were washed with PBS, incubated in the ABC reagent for 1 h at room temperature, washed again, and incubated in a peroxidase solution. The sections were then counterstained with hematoxylin, dehydrated, and coverslipped. Images were acquired using an Axioskop 2 plus microscope equipped with an AxioCam MRc5 CCD camera (Zeiss). Other samples were subjected to hematoxylin and eosin (H&E) staining for histological examinations.

Either HT29-MTX cells or ileum frozen sections were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized in 0.1% Triton X-100 in PBS for 5 min, and blocked in PBS containing 5% (v/v) normal goat serum (NGS) for 30 min at room temperature. Samples were then stained with primary antibody for overnight at 4 °C. Following three washes with PBS, the samples were incubated with Alexa 488-conjugated goat anti-rabbit/mouse IgM (Invitrogen), and counterstained with PI in PBS containing 5% (v/v) NGS for 2 h. After washing with PBS, samples were mounted on slides and visualized with an Olympus FluoView 300 confocal microscope (Melville, NY, USA) with x400 objective. For immunohistochemical analysis, ileum frozen tissues incubated with primary antibody for overnight at 4 °C were treated with biotinylated secondary antibody solution (Vectastain Elite ABC kit, Vector Laboratories, CA, USA) for 1 h at room temperature. Sections were washed with PBS, incubated in the ABC reagent for 1 h at room temperature, washed again and incubated in a peroxidase solution. Sections were then counterstained with hematoxylin, dehydrated, and coverslipped. Images were acquired using an Axioskop 2 plus microscope equipped with an AxioCam MRc5 CCD camera (Zeiss, Thornwood, NY, USA). Other samples were subjected to hematoxylin and eosin staining for histological examinations.

Cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized in 0.1% Triton X-100 in PBS for 5 min, and blocked in PBS containing 5% (v/v) normal goat serum (NGS) for 30 min at room temperature. Samples were then stained with primary antibody for overnight at 4 °C. Following three washes with PBS, the samples were incubated with Alexa 488-conjugated goat anti-rabbit/mouse IgM (Invitrogen Co., Carlsbad, CA, USA), and counterstained with PI in PBS containing 5% (v/v) NGS for 2 h. After washing with PBS, samples were mounted on slides and visualized with an Olympus FluoView™ 300 confocal microscope with 400x objective.