Heavy Metal Staining for TEM Thin Sections

Here is a protocol for staining thin sections for transmission electron microscopy:

Staining

Set up a moisture chamber:
• In an inverted 60 mm petridish place a 100 mm disk of filter paper and wet with ddH2O
• Cut a piece of parafilm to fit and place it on the wet filter paper in the dish.

Set of ddH2O wash:
• Cut a piece of parafilm
• Wet surface of bench with water
• Place film on water to stick to bench
• Place 100 uL drops of ddH2O on the film in an array of 3 x n where n = the number of grids to be stained

Staining:

UA:
• Place grid on a 5 uL drop of UA in a moisture chamber
• Cover with lid and place chamber in the dark or cover with metal foil to avoid light exposure to the uranium (light can cause U precipitate on section)
• Leave for 20 min.
• Pick up grid with tweezers and tap off remaining UA
• Immediately place onto the first 100 uL drop of ddH2O
• After 2 sec. move grid to second 100 uL drop and let sit for about 1 min.
• Transfer to the third drop for another minute
• Pick up grid with tweezers and tap off remaining water
• Transfer to grid box and allow grid to dry for several minutes

Pb:
• Cut a new piece of parafilm for the moisture chamber and place 4 or 5 pieces of Sodium Hydroxide around the edges and cover
• Place grid on 5 uL drop of lead citrate while avoiding any exhalation of breath(CO2 can cause Pb precipitate on section)
• Leave for no more than 5 min.
• Pick up grid with tweezers and tap off remaining Pb
• Immediately place onto the first 100 uL drop of ddH2O (again, be careful not to breath on the grid)
• After 2 sec. move grid to second 100 uL drop and let sit for about 1 min.
• Transfer to the third drop for another minute
• Pick up grid with tweezers and tap off remaining water
• Transfer to grid box and allow grid to dry for several minutes