Negative stain EM

Grids:
EMS FCF-300-Cu (a popular choice of ready-to-go, carbon-coated copper grids) [71 USD for a box of 50; Link ]

EMS G300-Cu (copper support grids, for making custom carbon-coated grids) [25 USD for a vial of 100; Link ] EMS 300-Cu-Rh (copper-rhodium support grids, allows for easier side identification as one surface is copper and one rhodium, for making custom grids) [25 USD for a vial of 100; Link ]

Note: To make custom grids, you need: a) access to a carbon evaporator machine, b) a compound for making a support layer for the carbon to rest on, such as EMS Collodion #12620 [16 USD for 30 ml; Link ]

Tweezers:
EMS Tweezer 0103-N4-PO (convenient choice for making EM grids since the tweezer holds a grid in place and only opens when depressed) [35 USD for one; purchase 2 pairs at least; Link ] Ted Pella Tweezer 525 (for manipulating grids) [58 USD for one; Link ] Ted Pella Tweezer 527 (for manipulating grids) [62 USD for one; Link ]

Stains:
EMS Phosphotungstic Acid #19500 [62 USD for 100 g; Link ] EMS Uranyl Acetate #22400 [162 USD for 125 g; Link ] EMS Uranyl Formate #22450 [102 USD for 1 g; Link ]

Other:
EMS grid boxes #71150 Whatman filter paper no. 1, 90 mm circles, #1001090 Parafilm

Uranyl Acetate 2%
Standard negative stain, good stability after preparation. pH of solution is 4 - 4.5.

50ml: 1. Weigh 1 g in 50 ml conical tube; 2. Add 50 ml ddH2O water; 3. Put on tube cap and shake until dissolved; 4. Filter through 0.22 μm filter into light-opaque brown glass bottle (or into opaque 50 ml conical tube); [alternatively, filter into regular 50 ml conical tube and wrap it in aluminum foil at the end]; 5. Store at 4oC, shelf-life: months; 6. If light stain precipitation is observed after a while, filter through 0.22 μm before use.

Uranyl Formate 0.75%
Fine grain size negative stain, recommended for small proteins < 100 kDa, poor stability after preparation. pH of solution is 4 - 4.5.

5ml: 1. Weigh out 37.5 mg of stain into a small beaker, add a stir bar; 2. Add 5 ml of boiling/very hot ddH2O water and stir vigorously for 15 minutes; 3. Add 8 μl of 5 M NaOH, continue stirring for another 15 minutes; 4. Filter through 0.22 μm filter into a 15 ml tube covered with aluminum foil (or 15 ml opaque tube); 5. Store at RT, shelf-life: up to a week (precipitation progresses quickly); 6. If traces of precipitation are visible, filter needed volume through 0.22 μm filter before use.

Phosphotungstic Acid 2%
Provides less contrast than uranyl acetate and formate, images tend to have a 'fuzzy' look to them, useful for work with specimen sensitive to low pH, pH of solution can be adjusted between 5 - 8.

20ml: 1. Weigh out 0.4 g of stain into a small beaker, add a stir bar; 2. Add 20 ml of ddH2O water and stir vigorously until dissolved; 3. Adjust the pH to anywhere between 5 - 8, with NaOH or KOH; 4. Filter through 0.22 μm filter into a 50 ml tube; 5. Aliquot 0.5 ml portions into 1. 5ml tubes and freeze at -20oC; 6. Break a fresh aliquot from freezer for staining of your samples.

Side blotting method
1. Glow-discharge grid at 25 mAmp for 45 sec. Do this directly before making grids as the applied charge dissipates over time; [This procedure modifies the carbon surface to more hydrophilic (negatively charged); alternatively it can be modified to a hydrophobic surface (positively charged) by the presence of amylamine during the process (put 2 drops of amylamine in a clean small cup, and place the cup in the glow-dicharger chamber). Glow-discharge also removes any residual hydrocarbons thus cleaning the carbon surface.] 2. On a piece of parafilm, place 3 x 8 ul drops of protein buffer and 3 x 8 ul drops of stain (2 for wash and 1 for stain); [The number of buffer washes and stain washes should be optimized for individual samples.] 3. Grip the edge of the grid with a pair of tweezers; 4. Apply 3.5 µL of sample to the carbon surface of the grid with a pipette; 5. Absorb the sample to the grid for 45 s; [This time should be optimized for individual samples, general range is 10-60 s.] 6. Blot away excess liquid by touching the edge of the grid to a piece of filter paper; 7. Wash grid with buffer: briefly touch the carbon surface of the grid to a buffer drop sitting on the parafilm and lift the grid, blot away excess liquid. Repeat the process 2 more times; 8. Wash with stain: perform 2 washes as described above; 9. Stain the sample: briefly touch the carbon surface of the grid to the last stain drop, lift it, stain for 45 s then blot away excess stain; [This time should be optimized for individual samples, typical times are 30 - 60 s.] 10. Air dry the grid for 3-5 min, then place in a storage box. [Ideally, storage boxes should be stored in a desiccator.]

Useful resources
1. Instructional video from the publication: Scarff, C. A., Fuller, M. J., Thompson, R. F., Iadaza, M. G. Variations on Negative Stain Electron Microscopy Methods: Tools for Tackling Challenging Systems. J. Vis. Exp. (132), e57199, doi:10.3791/57199 (2018).