Summary

Golgi in fixed and permeabilized NIH 3T3 mouse fibroblast cells was labeled with green-fluorescent Alexa Fluor® 488 conjugate of HPA lectin. Filamentous actin was stained with red-orange-fluorescent Alexa Fluor® 568 phalloidin. Mitochondria were labeled with a primary antibody directed against OxPhos Complex V Inhibitor Protein and visualized using far-red-fluorescent Alexa Fluor® 680 goat anti-mouse IgG. Nuclei were stained with blue-fluorescent DAPI.

Table 2 - Products Used

Methods and Materials

Tissue
BPAE Cells

Materials Needed

10X Phosphate-Buffered Saline (PBS) (GIBCO®)

Triton X-100 (Sigma)

Methanol-free 16% formaldehyde solution (Polysciences)

Unconjugated HPA lectin (E-Y Labs)

Alexa Fluor® 488 conjugate of HPA lectin (Molecular Probes®)

Heat-inactivated 100% Normal Goat Serum (NSG) (GIBCO®)

Bovine Serum Albumin (Sigma)

anti-OxPhos Complex V inhibitor protein, mouse IgG1, monoclonal 5E2 (anti-ATP synthase IP; anti-F1F0-ATPase IP) *human mitochondrial reactivity* (Molecular Probes®)

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

Note: Protocols for cell handling and general cell culture techniques can be found here.

The following protocol has been optimized for staining NIH 3T3 mouse fibroblast cells using 200 µl of staining solution at room temperature. Optimal staining conditions may vary slightly depending on sample conditions and should be determined for your sample.

Protocol

Solution Preparation
  1. To prepare 1 liter of 1X PBS solution, add 100 mL of 10X PBS and 900 mL of deionized water (dH2O) to a one liter container. For single assay preparation, add 1.0 mL of 10X PBS to 9.0 mL of dH2O to make 10 mL of 1X PBS. You will need 30 mL 1X PBS to make the fixative solution in the next step.
  2. To prepare a 4% fixative solution, transfer the entire contents of the ampule containing 16% formaldehyde solution into a separate container and add 30 mL of 1X PBS (prepared in step 1). We recommend a 50 mL centrifuge tube with a screw cap. Store unused 1X fixative solution at room temperature.
Sample Preparation
  1. Remove media from live cells and fix cells for 15 minutes in 4% formaldehyde solution.
  2. Wash 3 x 5 minutes in PBS.
  3. To reduce off-cell background caused by migration of the cells on the coverslip, incubate with 30 µg/ml unconjugated HPA lectin (diluted in PBS) for one hour.
  4. Wash 3 x 5 minutes in PBS.
  5. Permeabilize cells for 10 minutes with PBT (0.1% Triton X-100 in PBS).
  6. Block for 1 hour with blocking buffer (10% NGS and 1% BSA in PBT)./li>
Staining and Incubation
  1. Incubate cells with 5 µg/ml of anti-OxPhos Complex V inhibitor protein primary antibody (diluted in 1%BSA/PBT) for 60 minutes.
  2. Wash 3 x 10 minutes in PBT.
  3. Incubate cells with 5 µg/ml of Alexa Fluor® 680 goat anti–mouse IgG for 1 hour in 1% BSA/PBT.
  4. Wash 3 x 10 minutes in PBS.
  5. Label cells with a combined PBS solution of Alexa Fluor® 568 phalloidin (1:100 dilution from stock prepared above) and of Alexa Fluor® 488 HPA lectin conjugate (5 µg/ml) for 20 minutes.
  6. Wash 3 x 5 minutes in PBS.
  7. Label cells with 0.2 µg/ml of DAPI in PBS for 1 minute.
  8. Wash 3 x 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. 100x Oil 1.4 NA
Filters Used
DAPI Alexa Fluor® 488 Alexa Fluor® 568 Alexa Fluor® 680
Excitation: 330WB80 Excitation: 475AF40 Excitation: 535DF35 Excitation: 630AF50
Dichroic: 400DCLP Dichroic: 505DRLP Dichroic: 570DRLP Dichroic: 650DRLP
Emission: 450DF65 Emission: 535AF45 Emission: 590DF35 Emission: 695AF55
Imaging processing software: The MetaMorph Imaging System