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
1nm gold conjugated secondary antibody from Amersham. Dilute the
antibody 1:50 in washing buffer.
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
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.