Home › Immunology › Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids
Immunology JoVE (Open Access) Citable · DOI

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

DOI: 10.3791/56662-v
What you'll learn
  • ✓ Isolate 3D intestinal organoids from basement matrix cultures
  • ✓ Apply optimized fixation and immunolabeling protocols for microtubule and centrosomal proteins
  • ✓ Perform multi-marker immunofluorescence staining on intestinal tissue and organoids
  • ✓ Mount and visualize labeled organoids for microscopy analysis
Protocol

Biopharma Insights We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

Difficulty
intermediate
Total time
~4–6 hours per sample batch (fixation through mounting); organoid culture prior: 5–7 days
Model organism
Mouse intestinal organoids
Biosafety
BSL-1

Steps

1
Isolate intestinal organoids from basement matrix domes

Remove 24-well plates containing organoids embedded in basement matrix, aspirate growth medium, and mechanically isolate organoid structures from the surrounding matrix for downstream processing.

▶ 01:41
2
Fix isolated organoids with optimized fixation protocol

Apply appropriate fixation reagent to isolated organoids, incubate for specified duration to preserve cellular morphology and antigenicity for immunolabeling.

▶ 03:20
3
Block and apply primary and secondary antibodies

Incubate organoids in blocking solution to reduce non-specific antibody binding, then sequentially apply primary antibodies against microtubule, centrosomal, junctional, and stem cell markers, followed by fluorophore-conjugated secondary antibodies.

▶ 04:22
4
Mount labeled organoids on microscopy slides

Transfer immunolabeled organoids to glass slides using appropriate mounting medium with anti-fade reagent to preserve fluorescent signal and enable confocal or epifluorescence microscopy analysis.

▶ 06:41
5
Visualize immunolabeled organoid structures

Acquire and analyze multi-channel fluorescence images of mounted organoids to evaluate localization of microtubular networks, centrosomal structures, cell-cell junctions, and Lgr5+ stem cells.

▶ 07:39
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