Microscopy for 3D cultures

• Before starting
• Materials and supplies
• antibodies, stains and other reagents
• Possible panels
• Other consumables
• Fixing and permeabilizing cells
• Immunofluorescent staining
• Preparing for confocal microscopy
• 2D cultures
• LEICA SP5 Confocal Microscope Resources

Before starting

This protocol involves the use of the Leica SP5FLIM inverted microscope at PennVet, so you should receive training from Gordon Ruthel at the PennVet Microscopy Core before using this instrument. Microscopic images will be acquired using the Leica Application Suite Advanced Fluorescence (LAS AF) software. With the current excitation laser set-up on this microscope, an ideal 4-color experiment would be FITC/GFP, DAPI, Alexa 568 and Alexa 647.

Materials and supplies

antibodies, stains and other reagents

Possible panels

• UEA1-FITC, DAPI, ZO1-Alexa568, Ezrin-Alexa647
• C. rodentium-GFP, DAPI, Phalloidin-Alexa568, Ezrin-Alexa647

Other consumables

• 4% Paraformaldehyde (formaldehyde) aqueous solution (Electron Microscopy Sciences cat# 157-4-100)
• Aqueous solution of 10% Triton X-100 (Sigma, USA)
• Phosphate buffered saline – PBS (ice-cold for rinsing steps)
• Cell Imaging Dish, 170 μm thick, 35x10 mm (Eppendorf cat# 0030740017)
• Truncated P200 and P1000 pipette tips

Fixing and permeabilizing cells

• For details on recovering beads from STLV culture, see protocol here
• harvest a 1000μl aliquot of the the 3D beads Caco-2 cell culture using a CUT PIPPETE TIP and place it in an eppendorf tube
• Gently remove media with a pipette tip. No need to wash.
• Fix cells by adding 1ml of 4% paraformaldehyde. Gently but completely resuspend the beads.
• Incubate at room temperature for 10 min in fixative. Invert tubes periodically to resuspend beads
• Wash cells three times with ice-cold PBS. Incubate 5 min after each wash.
• Permeabilize cells by removing last PBS wash and adding 200μl of 0.25% Triton X-100.
• Incubate at room temperature for 10 min.
• Wash again 3x.
• The fixed and permeabilized bead preparation can be used immediately for staining or can be kept at 4 deg for use later.

Immunofluorescent staining

• Prepare ice bath with PBS
• Remove PBS from fixed/permeabilized cells
• Prepare ZO-1 (1/400) and Ezrin (1/400) antibodies by adding 2ul of each to 800ul of PBS. Scale up if more than 800ul is needed.
• Add enough antibody mix to cover beads
• Incubate for 1h at room temperature with occasional gentle mixing by inverting.
• Remove antibody and wash 3x with cold PBS allowing the beads to completely settle between washes
• Prepare UEA-FITC (1/400), secondary antibody conjugated to Alexa 568 (1/500), and secondary antibody conjugated to Alexa 647 (1/500) by adding 2.5ul, 2ul, and 2ul, respectively, in 1ml of PBS.
• Note If using phalloidin, dilute 1/400 for staining.
• Add enough reagent mix to cover cells.
• Incubate for 1h at room temperature with occasional gentle mixing by inverting.
• Remove antibody and wash 3x with cold PBS allowing the beads to completely settle between washes
• Cells are now ready to be mounted and imaged

Preparing for confocal microscopy

• Using a truncated pipette tip, carefully transfer 1-2 drops of the stained beads to a Cell Imaging Dish and add 1-2 drops of the Vectashield® antifade mounting medium and take it for microscopic observation. Protect from light (cover with alumnium foil) and heat (keep it on ice).

2D cultures

• If fixing/staining of 2D cultures is also needed, cells should be grown on Cell Imaging Dishes and fixed/stained in the same manner as 3D cultures above.

LEICA SP5 Confocal Microscope Resources

• User Manual
• SP5BasicModule YouTube video
• Leica SP5 Laser Scanning Confocal Microscope Training Session
• ThermoFisher Scientific Fluorescence SpectraViewer
• BTI: CONFOCAL LEICA SP5 user manual
• UMass: Leica TCS SP5 Confocal Laser Scanning Microscope User Guide
• Standard settings for Leica SP5 confocal microscope:

• Fluorophore Spectral data and corresponding laser for Excitation:

• Alexa Fluor Dyes: