7. Poststaining for electron microscopy
7A. Required materials:
KMnO 4
H 2 SO 4 1N
Uranyl acetate
Lead citrate
NaOH 2 N, carbonate free
7B. Preparations:
Acidified permanganate staining solution (0.1% KMnO 4 in 0.1 N H 2 SO 4
[49]): Prepare a stock solution of 1% KMnO 4 by adding 0.1 g KMnO 4 in 10 ml
of distilled water. Immediately before staining, add 100 lL of the KMnO 4 stock
solution and 100 lL of 1 N H 2 SO 4 to 800 lL of distilled water. The KMnO 4
stock solution can be stored for several months at room temperature.
5% aqueous uranyl acetate: Weigh 5 g of uranyl acetate under the fume hood
and add to 100 ml of ultrapure water. Cover the bottle with foil and dissolve
overnight on a shaker in a warm room (37 °C). Can be stored for several months
in the dark. Filter before use.
Lead citrate solution: Prepare fresh, during the uranyl acetate staining period.
Under the fume hood weigh out 0.01–0.04 g lead citrate in a 10 ml tube. Add
1 ml of carbonate free 2 N NaOH to dissolve lead and then add 9 ml of water.
Filter before using. The lead citrate powder can be weighed out in tubes ahead of
time and stored at room temperature for convenience.
7C. Procedures:
Dip coverslip with sections in a Coplin jar filled with ultrapure water (wet
sections stain better). Remove and put into petri dish with filter paper or gauze
underneath. Cover the sections with freshly made acidified permanganate
staining solution and leave for 1 min. Quickly remove stain and flood coverslip
with ultrapure water. Wash extensively with water.
Put coverslip in a clean petri dish and add a small amount of filtered 5% aqueous
uranyl acetate over the sections. Cover the petri dish and let stain for 30 min.
While coverslip is staining, make up fresh lead solution.
Remove UA staining solution and flood coverslip with water. Wash extensively
and then store in Coplin jar while getting lead stain ready. Add NaOH pellets to
staining dish.
Add freshly made and filtered lead citrate over the sections. Immediately close
the petri dish. Stain for 1 min ONLY! Remove lead and flood coverslip with
water. Wash extensively with water.
Let dry, then mount with carbon paint onto SEM stub.
Ribbons are imaged in an FESEM microscope using the backscatter detector at
5–8 keV.
178
K. D. Micheva and K. D. Phend
7A. Required materials:
KMnO 4
H 2 SO 4 1N
Uranyl acetate
Lead citrate
NaOH 2 N, carbonate free
7B. Preparations:
Acidified permanganate staining solution (0.1% KMnO 4 in 0.1 N H 2 SO 4
[49]): Prepare a stock solution of 1% KMnO 4 by adding 0.1 g KMnO 4 in 10 ml
of distilled water. Immediately before staining, add 100 lL of the KMnO 4 stock
solution and 100 lL of 1 N H 2 SO 4 to 800 lL of distilled water. The KMnO 4
stock solution can be stored for several months at room temperature.
5% aqueous uranyl acetate: Weigh 5 g of uranyl acetate under the fume hood
and add to 100 ml of ultrapure water. Cover the bottle with foil and dissolve
overnight on a shaker in a warm room (37 °C). Can be stored for several months
in the dark. Filter before use.
Lead citrate solution: Prepare fresh, during the uranyl acetate staining period.
Under the fume hood weigh out 0.01–0.04 g lead citrate in a 10 ml tube. Add
1 ml of carbonate free 2 N NaOH to dissolve lead and then add 9 ml of water.
Filter before using. The lead citrate powder can be weighed out in tubes ahead of
time and stored at room temperature for convenience.
7C. Procedures:
Dip coverslip with sections in a Coplin jar filled with ultrapure water (wet
sections stain better). Remove and put into petri dish with filter paper or gauze
underneath. Cover the sections with freshly made acidified permanganate
staining solution and leave for 1 min. Quickly remove stain and flood coverslip
with ultrapure water. Wash extensively with water.
Put coverslip in a clean petri dish and add a small amount of filtered 5% aqueous
uranyl acetate over the sections. Cover the petri dish and let stain for 30 min.
While coverslip is staining, make up fresh lead solution.
Remove UA staining solution and flood coverslip with water. Wash extensively
and then store in Coplin jar while getting lead stain ready. Add NaOH pellets to
staining dish.
Add freshly made and filtered lead citrate over the sections. Immediately close
the petri dish. Stain for 1 min ONLY! Remove lead and flood coverslip with
water. Wash extensively with water.
Let dry, then mount with carbon paint onto SEM stub.
Ribbons are imaged in an FESEM microscope using the backscatter detector at
5–8 keV.
178
K. D. Micheva and K. D. Phend