Skip to content

Latest commit

 

History

History
67 lines (60 loc) · 4.61 KB

Mucus_Staining.md

File metadata and controls

67 lines (60 loc) · 4.61 KB
Raw

Mucus thickness measurement assay

Background

The purpose of this experiment is to provide high-resolution quantification of gut microbiota spatial organization in the colon tissues. The mucus is highly transparent and not visible under normal conditions. Meanwhile, the high water content makes it sensitive to any methods that dries the mucus. In this protocol, we apply methacarn solution to preserve the mucus in histological sections, Fluorescence in situ hybridization (FISH) to detect bacteria, and immunostaining to identify mucins in the fixed tissue. The thickness of the mucus layer will be measured.

Materials

  • Mouse tissue dissection instruments
  • Container with methacarn solution (60% methanol, 30% chloroform, 10% acetic acid)
  • Paraffin embedding and tissue sectioning service
  • Oven, 50 and 60°C
  • Humidity chamber
  • PAP pen
  • Fluorescence microscope
  • Ethanol
  • Xylene
  • PBS
  • BSA Blocking Buffer, 5% in PBS (Thermo J61089-AP)
  • Eubacteria FISH probe - Cy3 (Sigma MBD0033)
  • Bacterial negative control FISH probe - Cy3 (Sigma MBD0035)
  • MUC2 Polyclonal Antibody, Invitrogen™ (Fisher PIPA5121758)
  • Anti-IgG Donkey Antibody (Alexa Fluor® 488) (VWR 102649-726)
  • Hybridization solution (20 mM Tris–HCl, pH 7.4, 0.9 M NaCl, 0.1% (w/v) SDS, 20% (v/v) formamide) heated to 50°C
  • FISH washing buffer (20 mM Tris–HCl, pH 7.4, 0.9 M NaCl)

Tissue Fixation to Preserve Intestinal Mucus

  • Mice are sacrificed according to the regulations. Minimize handling and avoid stressing the animals as intestinal emptying limits the sampling of the fecal-filled distal colon.
  • Dissect the intestine, cut pieces of the intestine containing fecal material (cut around the fecal pellets in the colon), and place them in a container with methacarn.
  • Fix the tissue for a minimum of 3 h and a maximum of 2 weeks at room temperature.
  • Wash in dry methanol for 30 min.
  • Wash in dry methanol for 30 min.
  • Wash in absolute ethanol for 20 min.
  • Wash in absolute ethanol for 20 min.
  • Incubate in xylene for 15 min.
  • Incubate in xylene for 15 min.
  • Tissue samples within cassettes are then submerged in melted paraffin at 68˚C for 2 h.
  • Remove samples and keep them at room temperature.
  • Paraffin embedding.
  • Paraffin blocks are cut into 4um-thick sections.
  • The sections are placed on glass after floating on a water bath as per standard procedures.
  • The embedded and sectioned material can be stored at room temperature.

Fluorescent In Situ Hybridization (FISH)

  • Dewax the sections by an initial incubation of the slides at 60°C for 10 min.
  • Pre-warm an empty Coplin jar, 100mL xylenes in a glass bottle at 60°C.
  • Incubate the slides in xylene substitute solution twice for 10 min.
  • Incubate the sections in 99.5% ethanol for 5 min and let the slides air dry.
  • Mix 0.36uL probe and 4.04uL hybridization solution and add to the slide (4.4uL/254mm2).
  • Overlay the liquid with a coverslip (18mm circle) and place the slide in a humid chamber. Incubate the sections at 50°C for 3 h.
  • Remove the cover glass and incubate slides in FISH washing buffer at 50°C for 10 min.
  • Wash the slide in PBS for 3× for 10 min.

Immunostaining

  • Mark around the sections with a PAP pen.
  • Add blocking buffer and incubate in darkness in a humidity chamber for 30 min at 4°C.
  • Dilute the polyclonal rabbit anti-mouse Muc2-specific antibody 1:200 in blocking buffer and add it to the slide (20uL/sample).
  • Incubate in darkness for 18 h at 4°C.
  • Wash the slide in PBS for 3× for 10 min.
  • Dilute the secondary antibody in blocking solution (Anti-rabbit Alexa 488, diluted 1:100 in blocking buffer) with 10ug/mL of DAPI, and add it to the sample (20uL/sample).
  • Incubate in the dark in a humid environment at 4°C for 2 h.
  • Wash the slide in PBS for 3× for 10 min.
  • Let the slide almost dry completely and mount the sections using Prolong antifade mounting media or glycerol and let it set at room temperature.
  • Analyze the sections using a fluorescent microscope.

References

  • Johansson ME, Hansson GC. Preservation of mucus in histological sections, immunostaining of mucins in fixed tissue, and localization of bacteria with FISH. Mucins: Methods and protocols. 2012:229-35.
  • Earle, Kristen A., Gabriel Billings, Michael Sigal, Joshua S. Lichtman, Gunnar C. Hansson, Joshua E. Elias, Manuel R. Amieva, Kerwyn Casey Huang, and Justin L. Sonnenburg. 2015. “Quantitative Imaging of Gut Microbiota Spatial Organization.” Cell Host & Microbe 18 (4): 478–88.