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Routine Transmission Electron Microscopy (TEM) Staining Protocol for Tissues
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Routine Transmission Electron Microscopy (TEM) Staining Protocol for Tissues

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 EMBed 812 and Propylene Oxide for1-2 hour.

10. 2:1 EMBed 812:Propylene Oxide overnight in dessicator with top off.

Embedding:

11. Embed in beam capsules.

12. Bake in 60 C oven for 48 hours.

Sectioning:

13. Cut thick sections (0.5-1.0 um), and use a steel loop to transfer the sections to a drop of water (this will flatten sections to avoid wrinkles - very important!!!) on slide and dry it on slide warmer or lamp. Toluidine blue stain for 2-5 min.

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

15. Ultrathin sections at 60-90 nm thick (silver-yellow color) and collect sections onto grids. Dry sections overnight before staining.

Staining:

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

17. Observe under electron microscope.

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 at 4 ºC. This solution is stable for at least 6 months at 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 ºC. Note: the mount of NaOH is very important. Too much will make solution cloudy.

EMBed 812:

Final VolumeEMBed-812DDSANMADMP-30 (2%)
20.91 ml10 ml4.5 ml6 ml0.41 ml
31.37 ml15 ml6.75 ml9 ml0.62 ml
41.82 ml20 ml9 ml12 ml0.82 ml
52.28 ml25 ml11.25 ml15 ml1.03 ml

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

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