Making Urynal Acetate and Lead Citrate

Reynolds lead citrate

Place the following components into a volumetric flask:

1.33 g Lead nitrate
1.76 g sodium citrate
30 ml ddH2O

• Shake vigorously for 1 min and then intermittently for the next 30 minutes. Add 8 ml of 1M NaOH, at which time the solution becomes clear. Use only freshly made carbonate-free NaOH solution. Add ddH2O to the 50 ml mark and mix by inversion until the precipitate is dissolved.
• The pH should be about 12. It will keep for several months if kept stoppered.
• Aliquot into 1.5 mL eppendorf tube through a hydrophobic filter.
• Centrifuge @12k RPM for 3.5 min. before staining.

Uranyl Acetate (3% in 50% Methanol)

Can be made in a 15 mL tube.

0.42 g UA
14 mL 50% Methanol

• Mix and dissolve on a rotator 30 min.
• Store for 2 days @4C before use
• Aliquot into 1.5 mL eppendorf tube through a hydrophilic filter.
• Centrifuge @12k RPM for 3.5 min. before staining.

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