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Author Topic: Immunogold-silver Labeling for EM (pre-embedding method)  (Read 5510 times)

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Offline ihcwor2

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Immunogold-silver Labeling for EM (pre-embedding method)
« on: March 12, 2003, 10:56:39 PM »
Immunogold-silver Labeling Protocol for Electron Microscopy (Pre-embedding Method)


Solutions and Reagents:

A.   0.2M Phosphate Buffer (0.2M PB):
To prepare 1 liter,
Na2HPO4, ----------------- 21.8 g.4 g
NaH2PO4 ----------------- 6.4 g
Distilled water ---------- 1000 ml
Mix to dissolve and adjust pH to 7.4

B.   0.1M Phosphate Buffer (0.1M PB):
To prepare 1 liter,
0.2M PB ------------------ 500 ml
Distilled water ----------- 500 ml

C.   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

D.   0.2M Citric Acid:
To prepare 100 ml,
Citric acid (monohydrate) --- 4.2 g
Distilled water ------------------ 100 ml

E.   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).

F.   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.

G.   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

H.   2% Glutaraldehyde in PBS:
To prepare 100 ml,
50% glutaraldyhyde -------- 4 ml
PBS ---------------------------- 100 ml

I.   1% Sodium Borohydrite Solution in 0.1M PB:
To prepare 100 ml,
Sodium borohydrite ----------- 1 g
0.1M PB ------------------------- 100 ml
             Stir to dissolve.

J.   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.

1.   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

K.   Primary Antibody:
Dilute in blocking buffer.

L.   Secondary Antibody:
1nm gold conjugated secondary antibody from Amersham. Dilute the antibody 1:50 in washing buffer.
                                                                                                       
M.   Silver Enhancement Solution:
Kit from Amersham
To prepare, mix equal drops of solution A and B before use.

N.   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.

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

P.   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.

Q.   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.


Procedure:

1.   Vibratome Section at 50 um thick.
2.   Collect sections in 0.1M PB.

3.   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.   Incubate in blocking buffer for 30 minutes.

7.   Primary antibody: incubate sections primary antibody in blocking buffer overnight at room temperature.
8.   Rinse in PBS for 3x5min.

9.   Incubate in washing buffer for 30 minutes.

10.   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.   Incubate sections in 2% glutaraldehyde in PBS for 10 minutes.
13.   Rinse 4x5 minutes in PBS.

14.   Silver enhancement:
a)   Rinse in 0.2M citrate buffer 3x2min.
b)   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).
c)   Rinse in 0.2M citrate buffer 2x5min
d)   Rinse in 0.1 M PB 4x5 min

15.   Post-fix in 1% OsO4 in 0.1M PB for 1 hour.
16.   Rinse in 0.1M PB for 3x5min.

17.   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.   Flat-embedding

23.   Bake 48 hours in 62 C oven.

24.   Trim the region of interest

25.   Cut ultrathin sections.

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

27.   Observation.

Immunogold-silver Labeling for EM (pre-embedding method)
« on: March 12, 2003, 10:56:39 PM »