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Author Topic: TEM-Cell Suspension/Cell Pellet Protocol  (Read 11729 times)

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Offline ihcwor2

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TEM-Cell Suspension/Cell Pellet Protocol
« on: March 12, 2003, 11:01:44 PM »
TEM-Cell Suspension/Cell Pellet 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 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:

6.   50% ethanol  15min
7.   70% Ethanol  15min
8.   95% Ethanol  15min
9.   100% Ethanol  2x15min
10.   100% Propylene oxide  2x15min
11.   1:1 Epon and Propylene Oxide for1-2 hour.
12.   2:1 Epon:Propylene Oxide overnight in dessicator with top off

Embedding:

13.   Embed in Beam capsules.
14.   60 C oven for 48 hours.

Sectioning:

15.   Semithin section and Toluidine blue stain for 2-5 min
16.   Observe sections under microscope for precise location to cut for ultrathin sections
17.   Ultrathin sections

Staining:

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

Observation:

14. Observe under electron microscope

TEM-Cell Suspension/Cell Pellet Protocol
« on: March 12, 2003, 11:01:44 PM »