Core Facilities
USF Health - College of Medicine

     

Light Microscopy

Sample slide preparation:
It is recommended to use #1.5 coverslip for sample preparation since most objective lenses are manufactured to be used with standard glass slides and coverslips of a certain thickness, usually 0.17 mm, which corresponds to the thickness of no. 1.5 coverslip.  
       
To prevent coverslip displacement and obtain the best images as possible, any excess amount of mounting medium should be removed after coverslip are placed. This can be easily done by placing a folded sheet of Kimwipes over the coverslip and gently tapping/pressing to squeeze out the excess medium until you get no excessive mounting medium. 
       
We also highly recommend that sample slides be sealed with nail polish or other sealing reagents. There are a couple of advantages of sealing; it helps to prevent samples from drying out, sliding of coverslip by the moving stages, embedded material movement during z-sectioning, and leaking of mounting medium and contaminating the immersion oil, which may blur your images and damage the lens. A cheap nail polish we recommend is the clear “Hard as Nails” from Sally Hansen available at grocery stores.

Preparation of glass coverslips for growing and imaging cells: 
For regular imaging, cells are plated on glass coverslip (12 mm circle or similar) inside of culture dish. The coverslips are typically pretreated with poly-L-lysine (PLL) if cells are loosely adherent to glass (polyornithine works better for some types of neurons).

Acid wash coverslips. This helps cells and polyamino acids stick to glass.
1. Heat coverslips in a loosely covered glass beaker in 1M HCl at 50-60 oC for 4-16h. 
2. Cool.
3. Wash coverslips extensively in dH2O, then ddH20.
4. Rinse coverslips in ethanol and leave to dry between a folded sheet of whatman paper(dry as separate coverslips).
5.Keep in a sterile tissue culture dish (can store for a year).     

Coat with polyamino acid.
1. Coat coverslips in bulk in 10-15ml 1mg/ml PLL (or 500mg/ml polyornithine), rocking or rotating for a minimum of 30 minutes in a 10 or 15cm tissue culture dish.
2. Save the polyamino acid (can reuse 3-4 times).

3. Wash the coverslips in dH2O, then ddH20 at least 5 changes in each (free polyaminoacid is cytotoxic). 
4. Rinse coverslips in 100% ethanol and dry those to be used immediately on one end in an open tissue culture dish in a sterile incubator. 
5. When dry, add cells.
6. Dry remaining coverslips between a folded sheet of whatman paper (dry as separate coverslips).
7. Keep in a sterile tissue culture dish (can store for a year). Do step 4 before use. Can keep 10-20 ml aliquots of 1mg/ml PLL and 500 mg/ml polyornithine stocks at -20 oC. High molecular weight PLL is standard (greater than 300K), but lower molecular weight PLLs can also be tried.

    

Optional-- Coat polyaminoacid/acid washed/coverslips with matrix molecules. This helps the attachment of very poorly adherent cells (e.g. neurons), and increases the growth rate of other cell types (e.g. primary culture cells). Different extracellular matrix molecules can also change the morphology of certain cell types (e.g protrusion of lamellipodia or filopodia, flattening of cell bodies useful for microinjection- usually determined empirically). 1. Coat individual polyamino acid/acid washed/coverslips with a drop of specific matrix molecule (held by surface tension) from frozen stocks for a minimum of 30 minutes at room temperature or in a 37 oC incubator, or overnight at 4 oC.
Examples:

          --Collagen type IV/PLL/acid washed coverslips

               PC12 cells (100ug/ml collagen), somites (2mg/ml collagen).

          --1x matrigel/PLL/acid washed coverslips

               Primary fibroblasts, neuroblastomas, fibromas, amphibian motor neurons,

               embryonic dorsal root ganglia.

          --10ug/ml laminin-polyornithine acid washed coverslips

               Adult dorsal root ganglia.
2. Wash 5x with calcium and magnesium free PBS, then 1x with culture media.

3. Plate cells. Coverslips must be coated fresh before plating cells. Washed, coated, coverslips can be stored for a maximum of one day in the cold room.

 

DNA (nuclear) staining:
Nucleus can be stained with various reagents that binds to nucleic acid. Some dyes (i.e. DAPI and Hoechst) bind preferentially to double-stranded DNA, whereas others interact with both DNA and RNA, which requires treatment of samples with RNase to enhance the DNA staining.

* DAPI (or Hoechst): This dye is convenient because of no need for pretreatment. Staining is normally performed after all other staining. Thus, if you are using Vectashield with DAPI mounting medium, just a drop of the medium takes care of DNA staining. 
Dilute the DAPI stock solution to 300 nM in PBS. Add this dilute solution just enough to cover cells on the coverslip preparation.  Incubate for 1–5 minutes and rinse the specimen several times in PBS. Mount the sample.

* Propidium Iodide and cyanine dyes (TO-PRO, YO-PRO....): Since these dyes bind well both DNA and RNA, sample need to be treated wiht RNAse for nuclei staining. During the secondary antibody staining, add 5 mg/ml of RNase A to the sample. Then, during final wash step, add the nucleic acid dye at ~0.5 mM and stain for 30 min to 1 hr at room temperature. Rinse well and mount the sample. 

Emission spectra of DNA-bound cyanine dimers (from Invitrogen).

7-AAD (7-aminoactinomycin D): This is a fluorescent dye that has a strong affinity to DNA by intercalating into GC-rich region. The unique feature of this dye is a very large Stokes shift (see figure); excitation at 546 nm and emission at 647 nm. It can be used directly to the sample without RNAse treatment.