Summary


Cytokeratin intermediate filaments in mouse trophoblast giant cells were labeled with a rat anti-cytokeratin monoclonal antibody and visualized using green-fluorescent Alexa Fluor® 488 goat anti-rat IgG. Nuclei were visualized with blue-fluorescent DAPI.

Methods and Materials


Cells

Trophoblast giant cells generated by Stealth RNAi silencing of OCT4 expression in cultured mouse embryonic stem cells

Materials Needed

KnockOut™ D-MEM (GIBCO®)

Leukemia inhibitory factor (LIF, ESGRO)(Chemicon)

KnockOut Serum Replacement (KSR)(GIBCO®)

Stealth RNAi duplexes designed to OCT4 (NM_013633) (Life Technologies™)

Lipofectamine™ 2000 transfection reagent (Life Technologies™)

Phosphate-Buffered Saline (PBS) 7.4 (10X), liquid (GIBCO®)

Methanol-free 16% formaldehyde solution (Polysciences)

Bovine serum albumin (Sigma)

Triton X-100 (Sigma)

rat anti-cytokeratin (University of Iowa Hybridoma Bank)

Alexa Fluor® 488 goat anti-rat IgG (Molecular Probes®)

Prolong® Gold antifade reagent (Molecular Probes®)

DAPI (Molecular Probes®)

Protocol


Sample Preparation

Mouse embryonic stem cells (mESC) cultured in serum-free medium (KnockOut™ D-MEM supplemented with L-glutamine, nonessential amino acids, -mercaptoethanol, penicillin/streptomycin, leukemia inhibitory factor (LIF, ESGRO) and 15% KnockOut™ Serum Replacement (KSR)) were transfected with Stealth™ RNAi and Lipofectamine™ 2000 transfection reagent. LIF was present in the media throughout cell culture and transfections. Transfected mESC were cultured out to 6 days.

Note: Information and protocols for the RNAi probe design and transfection can be found here.

Sample Preparation


  1. Remove media from live cells and fix cells for 15 minutes in 4% formaldehyde solution.

  2. Wash cells 3 x 5 minutes in PBS.

  3. Permeabilize cells by treating for 10 minutes with PBT (0.1% Triton X-100 in PBS).

  4. Wash cells 3 x 5 minutes in PBS.


Staining


  1. Block cells for 1 hour in antibody blocking buffer (10% normal goat serum (NGS), 1% BSA in PBT; PBT = PBS + 0.2% Triton X-100).

  2. Incubate cells with 5 µg/ml of anti-cytokeratin monoclonal antibody in antibody blocking buffer for 45 minutes.

  3. Wash cells 3 x 10 minutes in PBT.

  4. Incubate cells with 5 µg/ml Alexa Fluor® 488 goat anti-rat IgG in 1% BSA/PBT for 30 minutes.

  5. Wash 3 x 5 minutes in PBS.

  6. Incubate cells for 20 minutes with Alexa Fluor® 635 phalloidin, diluted 1:40 from stock solution.

  7. Wash 3 x 5 minutes in PBS.

  8. Label cells with 0.2 µg/ml DAPI in PBS for 1 minute.

  9. Wash 3 x 10 minutes in PBS.

  10. Mount the specimen in ProLong® Gold antifade reagent.


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.

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.

For more information on this experiment, check out the following reference:

Differentiation of mouse embryonic stem cells following RNAi-mediated silencing of OCT4 and Nanog

Shelley R. Hough, Ian Clements, Peter J. Welch, Kristin A. Wiederholt, Stem Cells First published online February 2, 2006 doi:doi:10.1634/Stem Cells Jun 2006; 24: 1467 - 1475

Table 1 - Instruments



Microscope Objective Used
Zeiss Axiovert 200M with CoolSnap HQ 12-bit digital camera 40x 1.2 NA water immersion
Filters Used
DAPI Alexa Fluor® 488
Excitation: 330WB80 Excitation: 475AF40
Dichroic: 400DCLP Dichroic: 505DRLP
Emission: 450DF65 Emission: 535AF45
Imaging processing software: The MetaMorph Imaging System

Table 2 - Products Used