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Author Topic: Immunogold-silver Labeling for EM (post-embedding method)  (Read 4970 times)

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Offline ihcwor2

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Immunogold-silver Labeling for EM (post-embedding method)
« on: March 12, 2003, 10:54:15 PM »
Immunogold-silver Labeling Protocol for Electron Microscopy (Post-embedding Method)


Solutions and Reagents:

A.   Phosphate Buffered (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.   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

C.   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.

D.   0.1M Glycine in 0.1M PB:
To prepare 100 ml, add 0.75 g of glycine to 100 ml of 0.1M PB.

E.   0.2M Sucrose Solution:
To prepare 100 ml, add 8 g of sucrose to 100 ml of 0.1M PB.    

F.   Post-fixative (1% Osmium Tetroxide in 0.1M PB):
To prepare 20 ml, add 5 ml of 4% osmium tetroxide to 5 ml of 0.2M PB, and then add 10 ml of 0.1M PB.

G.   Embed-812 Kit:
             Kit from EMS containing Embed-812, DDSA, NMA, and DMP-30.


H.   3% Hydrogen Peroxide in PBS:
To prepare 100 ml, add 10 ml of 30% H2O2 to 90 ml of PBS.

I.   Blocking Buffer (1% BSA, 3% NS, 0.1% Fish Gelatin in PBS):
To prepare 100 ml,
BSA ---------------------- 1 g
Normal serum --------- 3 ml
Fish gelatin ------------- 0.1 ml
PBS ---------------------- 100 ml
Stir to dissolve.

J.   Primary Antibody:
Dilution of primary antibody is critical for success of staining, so a titer must be performed at LM level prior to EM projects.

K.   Secondary Antibody:
An appropriate biotinylated secondary antibody should be selected and a titration must be performed at LM level prior to EM projects.

L.   10nm Gold Conjugated Streptavidin:
From EMS.

M.   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 in 4 C.

N.   Reynold's Lead Citrate Solution:
To prepare 50 ml, add 1.33 g of lead nitrate to 30 ml of distilled water. 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.


Procedure:

1.   Perfuse animals with fixative (preferred) or fix fresh tissue (about 1 mm3 in size) for overnight at 4 C.
2.   Rinse tissue samples in 0.1M PB for 3x10min.

3.   Rinse in 0.1M glycine in 0.1M PB to quench the free aldehyde groups.

4.   Incubate in 0.2M sucrose solution for 3x15min or overnight at 4C.

5.   Post-fix in 1% osmium tetroxide for 1 hour.
6.   Rinse in 0.1M PB briefly.

7.   Dehydrate through 50% ethanol, 70% ethanol, 95% ethanol for 10 minutes each
8.   Incubate in 100% ethanol for 2x15min.
9.   Incubate in 100% propylene oxide for 2x15min.
10.   1:1 Embed-812 and propylene oxide for overnight at room temperature

11.   Embed in beam capsules with straight Embed-812
12.   Bake in 62 C oven for 24 hours.

13.   Trim blocks, cut semithin sections at 1um thick, and stain with toluidine blue.
14.   Select region of interest and trim blocks to an appropriate size.

15.   Cut ultrathin sections.
16.   Collect sections on Nickel Grids (Grids need to be cleaned in acetone prior to use).
17.   Let the grids dry overnight prior to staining.
18.   Rinse grids in PBS briefly.

19.   Pretreatment: Incubate sections on droplets of 3% H2O2 for 10 minutes (Soften resin sections and remove OsO4).
20.   Rinse sections on large drops of PBS for 2x5min.
 
21.   Incubate sections in blocking buffer for 30 minutes.
22.   Rinse on large drops of PBS for 2x5 min.

23.   Incubate sections on droplets of primary antibody diluted in blocking buffer for 2 hours at room temperature or overnight at 4 C. Note: 1) Primary antibody dilution should be about 2 times more concentrated than LM working dilution. 2) Overnight incubation seemed to be better than 2 hours in room temperature.
24.   Rinse on large drops of PBS for 6x5 minutes.

25.   Incubate grids on droplets of biotinylated secondary antibody diluted in blocking buffer for 30 minutes. Note: We usually use secondary antibody (1:200, 2 times more concentrated than LM working dilution) from Vector Labs.
26.   Rinse on large drops of PBS for 6x5 minutes.

27.   Incubate sections with gold conjugated streptavidin (EMS, 10nm gold) diluted with PBS (1:20) for 30 minutes.
28.   Rinse on large drops of PBS for 6x5 minutes.

29.   (If doing double labeling, you need to repeat from step 21 to 28 (A different blocking buffer may be needed depending on species of 2nd antibody. Step 21 is not necessary if the species of 2nd antibody are the same as previous one). Note: For double labeling, use small gold conjugated streptavidin (i.e. 6nm) first, and use large gold conjugated streptavidin (i.e. 10nm) last.
30.   Rinse 3x10 dips in distilled water.

31.   Stain with uranyl acetate for 15 minutes and lead citrate for 1-2 minutes.
32.   Observe sections under electron microscopy.

Immunogold-silver Labeling for EM (post-embedding method)
« on: March 12, 2003, 10:54:15 PM »