Routine TEM Staining Protocol for Cells

 

Fixation:

 

1.       Fix cell suspension or free cells in 4% formaldehyde and 1% glutaraldehyde in 0.1 M PB (pH 7.4) by mixing equal volume of fixative and cell suspension.

2.       Transfer cells to centrifuge tube and spin for 10 minutes. A nice, tight pellet will be formed (Re-spin if necessary during processing). Carefully pipette off fixative. Add fresh fixative for at least 2 hours or overnight.

3.       Pipette off fixative. Replace with 8% (0.2M) sucrose in 0.1 M PB 3x15 minutes or overnight at 4 C (Samples can be kept in sucrose for a long time).

4.       Post-fix with 1% OsO₄ in 0.1 M PB for 1 hour.

5.       Pipette off OsO₄, Rinse in 0.1 M PB 3x10 minutes.

 

Dehydration:

 

1.       50% ethanol  15min

2.       70% Ethanol  15min

3.       95% Ethanol  15min

4.       100% Ethanol  2x15min

5.       100% Propylene oxide  2x15min

6.       1:1 Epon and Propylene Oxide for1-2 hour.

7.       2:1 Epon:Propylene Oxide overnight in dessicator with top off.

 

Embedding:

 

8.       Embed in Beam capsules.

9.       60 C oven for 48 hours.

 

Sectioning:

 

10.    Semithin section and Toluidine blue stain for 2-5 min

11.    Observe sections under microscope for precise location to cut for ultrathin sections

12.    Ultrathin sections

 

Staining:

 

13.    Uranyl acetate and lead citrate

 

Solutions and Reagents:

 

0.2M PB, pH 7.4:

Na₂HPO4 ------------------------ 21.8 g

NaH₂PO4 ------------------------ 6.4 g

Distilled water ----------------- 1000 ml

 

4F1G Fixative (4% Formaldehyde & 1% Glutaraldehyde in 0.1 M PB, pH 7.4):

0.1M PB, pH 7.4 --------------- 88 ml

37-40% Formaldehyde ----- 10 ml

50% Glutaraldehyde --------- 2 ml  

 

8% (0.2M) Sucrose in 0.1M PB:

Sucrose ------------------------- 8 g

0.1 M PB ------------------------ 100 ml

 

1% Osmium in 0.1M PB:

2% Osmium -------------------- 5 ml

0.2M PB, pH7.4 ---------------- 5 ml

 

5% Uranyl Acetate Solution:

To prepare 50 ml, add 2.5 g of uranyl acetate to 50 ml of distilled water. Cover with foil and stir overnight. Add 10 drops of glacial acetic acid. Store solution in 4 C.

 

Reynold’s Lead Citrate Solution:

To prepare 50 ml, add chemicals in distilled water in following order

Lead nitrate ------------------------------- 1.33 g

Sodium citrate, dihydrate -------------- 1.76 g (solution becomes cloudy when sodium citrate is added)

1N NaOH ----------------------------------- 5 ml  (solution becomes clear when NaOH is added)

Distilled water ----------------------------- 30 ml

Stir for 10 minutes to dissolve and add additional 15 ml of distilled water. Store solution for 3-6 months at 4 C. Note: the mount of NaOH is very important. Too much will make solution cloudy.

 

Epon 812:

 

Final Volume	EM bed-812	DDSA	NMA	DMP-30 (2%)
20.91 ml	10 ml	4.5 ml	6 ml	0.41 ml
31.37 ml	15 ml	6.75 ml	9 ml	0.62 ml
41.82 ml	20 ml	9 ml	12 ml	0.82 ml
52.28 ml	25 ml	11.25 ml	15 ml	1.03 ml

          Mix all four components in plastic beaker and stir with wood stick.