EM Immunogold Labeling Protocol - Pre-embedding Method
Procedure:
1. Sectioning: Vibratome sections at 50 um thick.
2. Collect sections in 0.1M PB.
3. Pretreatment: treat sections with 1% sodium borohydride solution for 30 minutes.
4. Rinse many times in 0.1M PB to remove bubbles.
5. Rinse in PBS for 3x5min
6. Serum Blocking: incubate in blocking buffer for 30 minutes.
7. Primary Antibody: incubate sections with primary antibody (appropriate diluted in blocking buffer) overnight at room temperature.
8. Rinse in PBS for 3x5min.
9. Washing Buffer: incubate in washing buffer for 30 minutes.
10. Secondary Antibody: incubate sections in gold-conjugated secondary antibody diluted 1:50 in washing buffer for 3 hours.
11. Rinse 4x5 min in washing buffer and then 4x5 min in PBS.
12. Post-fixation1: fix sections with 2% glutaraldehyde in PBS for 10 minutes.
13. Rinse 4x5 minutes in PBS.
14. Silver enhancement:
· Rinse in 0.2M citrate buffer 3x2min.
· Incubate in silver enhancement solution for 3-9 minutes for EM and 6-12 minutes for LM (If room temperature is very low, a longer time may be needed such as 20-30 min).
· Rinse in 0.2M citrate buffer 2x5min
· Rinse in 0.1 M PB 4x5 min
15. Post-fixation2: fix sections with 1% OsO4 in 0.1M PB for 1 hour.
16. Rinse in 0.1M PB for 3x5min.
17. Dehydration: dehydrate in 50%, 70% and 95% ethanol for 10 minutes each.
18. 100% ethanol 2x15 min.
19. Propylene oxide 2x15 min.
20. Incubate sections in 1:1 mixture of Embed-812 & propylene oxide for overnight.
21. Transfer sections to straight Embed-812 for 2 hours.
22. Embedding: flat-embedding, and bake in 62 C oven for 48 hours.
23. Sectioning: choose the region of interest, cut off and glue on blank flat-end beam capsule using super glue. Bake to dry for 30 minutes to 1 hour. Trim and cut ultrathin sections at 60-90 nm.
24. Contrast Staining: stain sections with uranyl acetate for 15 minutes and lead citrate for 5 minutes.
27. Observation.
Solutions and Reagents:
0.2M Phosphate Buffer (0.2M PB):
To prepare 1 liter,
Na2HPO4, -------------------- 21.8 g
NaH2PO4 --------------------- 6.4 g
Distilled water ------------- 1000 ml
Mix to dissolve and adjust pH to 7.4
0.1M Phosphate Buffer (0.1M PB):
To prepare 1 liter,
0.2M PB --------------------- 500 ml
Distilled water -------------- 500 ml
10X Phosphate Buffered Saline (PBS):
To prepare 1 liter,
Na2HPO4 --------------------- 10.9 g
NaH2PO4 --------------------- 3.2 g
NaCl --------------------------- 90 g
Distilled water -------------- 1000 ml
Mix to dissolve and adjust pH to 7.4
0.2M Citric Acid:
To prepare 100 ml,
Citric acid (monohydrate) ------ 4.2 g
Distilled water -------------------- 100 ml
0.2M Sodium Citrate Buffer (fresh):
To prepare 100 ml,
Sodium citrate (dihydrate) ------ 5.88 g
Distilled water --------------------- 100 ml
Adjust pH to 7.4 with 0.2M citric acid (keeps refrigerated).
Fixative (4% Paraformaldehyde and 0.5% Glutaraldehyde in 0.1M PB):
To prepare 1 liter,
Paraformaldedyde -------------- 40 g
0.1M PB --------------------------- 1000 ml
Heat to 65 C. Add a few drops of 1N NaOH to clear the solution. Stir to dissolve. Filter the solution and add 10 ml of 50% glutaraldehyde.
Post-fixative (1% Osmium Tetroxide in 0.1M PB):
To prepare 20 ml,
4% osmium tetroxide ---------- 5 ml
0.2M PB --------------------------- 5 ml
0.1M PB -------------------------- 10 ml
2% Glutaraldehyde in PBS:
To prepare 100 ml,
50% glutaraldyhyde ------------ 4 ml
PBS -------------------------------- 100 ml
1% Sodium Borohydrite Solution in 0.1M PB:
To prepare 100 ml,
Sodium borohydrite -------------- 1 g
0.1M PB ---------------------------- 100 ml
Stir to dissolve.
Blocking Buffer (1% BSA, 3% NGS, 0.04% Triton in PBS):
To prepare 100 ml,
Triton X-100 ----------------------- 0.04 ml
BSA ---------------------------------- 1 g
Normal goat serum -------------- 3 ml
PBS ---------------------------------- 100 ml
Stir to dissolve.
Washing Buffer (0.8% BSA, 0.1% Fish Gelatin in PBS).
To prepare 100 ml,
BSA ---------------------------------- 0.8 g
Fish gelatin ------------------------ 0.1 ml
PBS --------------------------------- 100 ml
Secondary Antibody:
1nm gold conjugated secondary antibody from Amersham. Dilute the antibody 1:50 in washing buffer.
Silver Enhancement Solution:
Kit from Amersham and prepare solution according to the instruction provided.
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.
1:1 Mixture of Embed-812 & Propylene Oxide:
To prepare 20 ml,
Embed-812 ------------------------- 10 ml
Propylene oxide ------------------- 10 ml
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.
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.
|
