Methods and Materials
- Phosphate-buffered saline (PBS) (Gibco)
- Triton X-100 (Sigma)
- BrainStain Imaging Kit (Invitrogen) containing:
- FluoroMyelin Green fluorescent myelin stain, 1 ml, 300X solution in water
- NeuroTrace 530/615 red fluorescent Nissl stain, 1 ml, 300X solution in DMSO
- DAPI dihydrochloride, 1 ml, 300X solution in water
- ProLong Gold antifade reagent *special packaging* (Invitrogen)
The following protocol has been optimized for staining 12 µm mouse brain 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. This protocol combines all three stains into one staining.
- For imaging, bring the tissue sections on slides to room temperature.
- Rehydrate and permeabilize sections in PBT (PBS + 0.2% TRITON X-100) for at least 20 minutes.
Staining Solution Preparation and Incubation
- Prepare the three-color BrainStain staining solution just prior to staining the section by diluting each stock solution 300-fold into PBS in a single vial. For example, to prepare enough staining solution for 5 slides (1 mL), add 3.3 µl of each stain to 990 µl PBS.
- Flood the sections with 200 µl staining solution; Protect the sample from light and incubate for 20 minutes at room temperature.
- Rinse with PBT; wash 3 x 10 minutes with PBT. To increase the specificity of the NeuroTrace stain, an extra wash or a longer wash (up to 2 hours) in PBT can be used.
Mounting Reagent Preparation and Sample Processing
- 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.
- 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.
- 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.
Table 1 - Instruments
|Nikon Eclipse 800 with Micromax 1300 YHS 12 bit digital camera.||40x 0.15 NA|
|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
Cytokeratin and nuclei in mouse trophoblast giant cells
α - catenin in HeLa human cervical cancer cells
Golgi, f-actin, nuclei, and mitochondrial OxPhos Complex V Inhibitor Protein in NIH 3T3 cells
Goblet and Paneth cells, f-actin, and nuclei in mouse intestine
For Research Use Only. Not for use in diagnostic procedures.