Summary

Mucus of goblet and Paneth cells in a 15 µm mouse intestine cryosection was stained with blue-fluorescent Alexa Fluor® 350 wheat germ agglutinin. Filamentous actin prevalent in the brush border and smooth muscle layer was stained with red-orange–fluorescent Alexa Fluor® 568 phalloidin. Nuclei were stained with SYTOX® Green nucleic acid stain.

Methods and Materials


Phosphate-Buffered Saline (PBS) (GIBCO®)

Triton X-100 (Sigma)

Bovine Serum Albumin (BSA) (Sigma)

Alexa Fluor® 350 wheat germ agglutinin (AF350 WGA) (Molecular Probes®)

Alexa Fluor® 568 phalloidin (Molecular Probes®)

RNAse (Sigma)

SYTOX® Green nucleic acid stain (Molecular Probes®)

ProLong® Gold antifade reagent *special packaging* (Molecular Probes®)

Note: Protocols for harvesting, embedding and preparing tissue sections can be found at "Links To Protocols"

The following protocol has been optimized for staining 15 µm mouse intestine cryosections on Superfrost Plus slides (Erie Scientific Co.), using 200 µl of staining solution within a PAP pen (Polysciences, Inc.) well at room temperature. Optimal staining conditions may vary slightly depending on sample conditions and should be determined for your sample.

Protocol

Sample Preparation

  1. For imaging, bring the tissue sections on slides to room temperature.
  2. Rehydrate in PBS for 30 minutes.
  3. Permeabilize sections in PBT (PBS + 0.2% TRITON X-100) for at least 10 minutes.
  4. Rinse twice in PBS for 5 minutes.
  5. Transfer sections into 1% BSA in PBS for at least 45 minutes.

Staining Solution Preparation and Incubation

Alexa Fluor® 350 Wheat Germ Agglutinin (WGA)

  1. Dissolve the Alexa Fluor® 350 WGA (5 mg) in 5 mL PBS for a final concentration of 1 mg/mL. This can be stored at 2–6ºC if sodium azide is added at a final concentration of 2 mM. Otherwise, the solution should be aliquoted and stored at –20ºC.
  2. Prepare a fresh stock solution of Alexa Fluor® 350 WGA in PBS at a final concentration of 0.5 mg/mL. For example, to stain 5 slides, dilute 500 µL of the stock solution with 500 µL of PBS. Centrifuge for 10 minutes at 10,000 rpm to remove any undissolved aggregates.
  3. Flood the sections with 200 µl staining solution; protect the sample from light and incubate for 30 minutes at room temperature.
  4. Wash 2-10 minutes in PBS.

Alexa Fluor® 568 phalloidin

  1. Dissolve 300 U of Alexa Fluor® 568 Phalloidin into 1.5 mL methanol to prepare a stock solution of 200 units/mL. Store at 2–6ºC.
  2. Prepare a fresh solution by diluting the stock solution 1:25 in PBS. For example, to stain 5 slides, add 40 µL of this stock solution into 960 µL of PBS.
  3. Flood the sections with 200 µl of staining solution; protect the sample from light and incubate for 30 minutes at room temperature.
  4. Wash 2-10 minutes in PBS.

SYTOX® Green nucleic acid stain

It is recommended that the sections be pretreated by RNAse digestion. This may help reduce nonspecific cytosolic staining when using SYTOX® Green stain.

  1. Prepare the RNAse digestion solution at a final concentration of 10 µg/mL in PBS.
  2. Incubate the tissue for 2 hours at 37 C.
  3. Wash 3-5 minutes in PBS.
  4. Prepare a fresh staining solution at a final concentration of 2.5 µM by diluting 1 µL of SYTOX® Green nucleic acid stain in 2 mL distilled, deionized H2O.
  5. Stain for 20 minutes at room temperature.
  6. Wash 3-10 minutes in PBS.

Mounting Reagent Preparation and Sample Processing

  1. Remove the ProLong® Gold antifade reagent from the freezer and allow the vial to equilibrate to room temperature. Using an external heat source to warm the vial is not recommended, as this may decrease the long-term stability of the product.
  2. Remove any excess liquid from the specimen and apply 1 or 2 drops (depending on the surface area of your sample) of the antifade reagent to the specimen. Cover slide-mounted specimens with a coverslip; for specimens mounted on coverslips, place a drop of antifade reagent onto a clean slide and carefully lower the coverslip onto the antifade reagent to avoid trapping any air bubbles.
  3. Allow the mounted sample to cure on a flat surface in the dark. Curing time may vary from a couple of hours to overnight, depending on the thickness of the sample and the relative humidity of the surrounding air. For long-term storage, seal the coverslip to the slide after curing to prevent excessive shrinkage of the mounting medium, which can result in sample distortion. After sealing, store the slide upright in a covered slide box at <=–20°C. Desiccant may be added to the box to ensure that the slide remains dry.

To view the samples immediately, secure the coverslip at the corners using nail polish or hot wax to prevent the coverslip from moving. Leave the edges clear to allow the preparation to cure.


Note: The antifade properties of ProLong® Gold antifade reagent improve slightly the longer it remains in contact with the specimen. To further reduce photobleaching, minimize the exposure of fluorescently labeled specimens to light by using neutral density filters, and expose samples only when observing or recording a signal. Optimize image capture by using a minimum of optics, high–numerical aperture objectives, relatively low magnification, high quality optical filters, and high-speed film or high-efficiency detectors.

Table 1 - Instruments



Microscope Objective Used
Nikon Eclipse 800 with MicroMax 1300 YHS 12-bit digital camera 40x 0.75 NA
Filters Used
Alexa Fluor® 350 WGA WGA SYTOX® Green Alexa Fluor® 568 phalloidin
Excitation: 330WB80 Excitation: 475AF40 Excitation: 535DF35
Dichroic: 400DCLP Dichroic: 505DRLP Dichroic: 570DRLP
Emission: 450DF65 Emission: 535AF45 Emission: 590DF35
Imaging processing software: The MetaMorph Imaging System

Table 2 - Products Used

Sku Name Size Price Qty
S7020 SYTOX® Green Nucleic Acid Stain - 5 mM Solution in DMSO 250 µL USD 201.00
W11263 Wheat Germ Agglutinin, Alexa Fluor® 350 Conjugate 5 mg USD 275.00
A12380 Alexa Fluor® 568 Phalloidin 300 units USD 373.00
F24631 FluoCells® Prepared Slide #4 (mouse intestine section with Alexa Fluor® 350 WGA, Alexa Fluor® 568 Phalloidin, and SYTOX® Green) 1 each USD 191.00
P36934 ProLong® Gold Antifade Reagent 5 x 2 mL USD 159.00