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Author Topic: Routine TEM Protocol  (Read 5127 times)

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Offline ihcwor2

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Routine TEM Protocol
« on: March 12, 2003, 11:00:21 PM »
Routine TEM Protocol


Solutions and Reagents:

A.   0.2M PB, pH 7.4:
                Na2HPO4 --------------- 21.8 g
                NaH2PO4 --------------- 6.4 g
                Distilled water --------- 1000 ml

B.   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

C.   8% (0.2M) sucrose in 0.1 M PB:
                Sucrose ------------------------- 8 g
                0.1 M PB ----------------------- 100 ml

      D.  1% Osmium in 0.1 M PB:
                2% Osmium -------------------- 5 ml
                0.2M PB, pH7.4 --------------- 5 ml

E. Embed-812:
Embed-812 Kit from EMS containing Embed-812, DDSA, NMA, and DMP-30. Make Embed-812 according to instruction provided with the kit.

F. 1:1 Mixture of Embed-812 & Propylene Oxide:
To prepare 20 ml,
Embed-812 ------------------------ 10 ml
Propylene oxide ------------------ 10 ml

G. 5% Uranyl Acetate Solution:
To prepare 50 ml,
Uranyl acetate --------------------- 2.5 g
Distilled water ---------------------- 50 ml
Cover with foil and stir overnight.
Add 10 drops of glacial acetic acid and Store solution in 4 C for 3 month.

H. Reynold's Lead Citrate Solution:
To prepare 50 ml,
Lead nitrate ------------------------ 1.33 g
Distilled water --------------------- 30 ml
Stir to dissolve.
Add 1.76 g of sodium citrate dihydrate and stir for 30 min
Add 8 ml of 1N NaOH and 12 ml of distilled water
Store solution in 4 C for three month.

   
Fixation:

1.   Fix 1 mm tissue blocks in 4% formaldehyde and 1% glutaraldehyde in 0.1 M PB (pH 7.4) for at least 2 hours to overnight.
2.   Immerse in 8% (0.2M) sucrose in 0.1 M PB 3x15min or overnight.
3.   Post-fix in 1% osmium tetroxide in 0.1 M PB 1 hour.


Dehydration:

4.   50% ethanol  15min
5.   70% Ethanol  15min
6.   95% Ethanol  15min
7.   100% Ethanol  2x15min
8.   100% Propylene oxide  2x15min
9.   1:1 Epon and Propylene Oxide for1-2 hour.
10.   2:1 Epon:Propylene Oxide overnight in dessicator with top off


Embedding:

11.   Embed in Beam capsules.
12.   60 C oven for 48 hours.

Sectioning:

13.   Semithin section at 1 um and Toluidine blue stain for 2-5 min
14.   Observe sections under microscope for precise location to cut for ultrathin sections
15.   Ultrathin sectioning

Staining:

16.   Stain with uranyl acetate for 15 minutes and lead citrate for 2-5 minutes.

Observation:

17. Observe under electron microscope

Routine TEM Protocol
« on: March 12, 2003, 11:00:21 PM »