Immunoperoxidase Labeling – TEM
Labeling antigens for electron microscopy (EM) presents many obstacles in having to compromise between antigen stability and ultrastructural preservation.
Immuno-labeling prior to embedding for accessible antigens allows for mild pre-fixation conditions followed by immune reaction and subsequent use of stronger fixatives to optimize ultrastructure. An additional advantage is that reactions can be confirmed by light or fluorescent microscopy prior to the time-consuming processes of embedding and sectioning.
(Adapted from Farquhar et al., Meth Cell Biol., Vol. 31, pp. 567)
- Hela 2 x 105/ml on thermonox coverslips in 24 well plates - Infect as appropriate
- Rinse 2X w/Hank’s buffered saline solution (HBSS)
Caution: For all fluid exchanges, remove most of the liquid but not all to avoid drying of sample which adversely affects the ultrastructure.
- Fix with PLP fixative (see below) + 0.25% glutaraldehyde
(Titrate glutaraldehyde for each antigen/antibody. If necessary, glutaraldehyde can be omitted, although te use of glutaraldehyde improves ultrastructural preservation)
- Rinse 3X w/ PBS (50 mM NaPO4/150 mM NaCl; pH 7.4)
(Commercial PBS will work if it doesn’t have any additives like azide)
- Permeabilize in PBS + 0.01% saponin. (titrate saponin; range = 0.005 - 0.05) - 5 min @ RT
(Make fresh daily from powder) Include this saponin concentration for all antibody steps)
- Add primary antibody in 1XPBS + 0.01% saponin - 1 h @ room temperature (RT )
- Rinse 3X w/ PBS @ RT
- Add secondary antibody: FAb -ï€¢-mIgG-HRP 1:100 in PBS + 0.01% saponin - 1 h @ RT
(Choose appropriate host specific HRP conjugated antibody)
- Rinse 3X w/ 1XPBS – @ RT
- Fix in 1.5% glutaraldehyde in 0.1 M sodium cacodylate + 5% sucrose; pH 7.4 -1 h @ RT
- Rinse 3X w/ 50 mM Tris-HCl + 7.5% sucrose; pH 7.4
- Add Enhanced DAB reagent (e.g., Pierce) < order fresh monthly/keep frozen until just prior to use 15 minutes -1 hour @ RT or until brown color becomes visible.
- Rinse 3X5 minutes w/ 50 mM Tris-HCl + 7.5% sucrose; pH 7.4
- Final fix with 2.5% glutaraldehyde in 0.1 M sodium cacodylate ; pH 7.4
- Rinse 3X5 minutes with 0.1 M sodium cacodylate; pH 7.4
- Post fix for 15 minutes – 1 hr with 1.0% osmium tetroxide/0.8% potassium ferricyanide in sodium cacodylate
- Rinse 1X5 minutes with sodium cacodylate
- Rinse 2X5 minutes with dH2O
- Dehydrate with a graded ethanol series, e.g. 50%, 75%, 95%, 3X100% for 5 minutes each.
- Infiltrate and embed with resin such as Spurr’s.
Note: Beem capsules can be filled with resin, and coverslips (after a minimum of one hour infiltration with 100 percent resin, can be inverted so the cell layer side is down on the top of a capsule. After curing, the Beem capsule can be dipped for 5-10 seconds in LN2, and the coverslip knocked off exposing the cells which should adhere to the resin surface of the Beem capsule. Alternatively, many cells can be grown on aclar which can be peeled off the beem capsule after curing.
- 16% Paraformaldehyde (formaldehyde) aqueous solution
- 8% Glutaraldehyde Aqueous Solution
- Code number 111-036-045, Peroxidase-AffiniPure F(ab')2 Fragment Goat Anti-Rabbit IgG (H+L) (min X Hu Sr Prot)
- Code number 115-036-062, Peroxidase AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG (H+L) (min X Hu,Bov,Hrs Sr Prot)
LAMP antibody (control), e.g., The Developmental Studies Hybridoma Bank, H4A3
Once you have determined the ideal concentration of paraformaldehyde (PFA) and glutaraldehyde (Glut) fixatives on each antigen/antibody, prepare the fixatives for EM as below.
- PLP Fixative
Soln. A: 0.1 M lysine HCl - NaPO4 buffer 7.5 ml Soln. B: 8% paraformaldehyde 2.5 ml Sodium meta periodate 21.3 mg 25% Glutaraldehyde 100 µl
McLean and Nakane PLP fixative: (From Farquhar et al., Meth in Cell Biol, Vol 31, pp. 567)
- Solution A: 0.1 M lysine – sodium phosphate buffer
- Add 1.83 g lysine HCl to 50 ml dH2O
- Add 0.1 M Na2HPO4 until pH 7.4 (~5 mls)
- Bring up to 100 ml with 0.1 M NaPO4, pH 7.4 buffer.
- Prepare this freshly
- Solution B: 8% paraformaldehyde
- Add 8 grams paraformaldehyde (Fisher Scientific to ~95 mls of dH2O.
- Heat to 60° C with stirring.
- Add 1N NaOH dropwise until solution clears
- Bring up to 100 ml with dH2O. Filter
- Keep frozen at –70 C until ready to use
- Prepare 8% paraformaldehyde from 16 % stocks solution by diluting it in distilled water
- Mix three parts solution A with one part solution B, and add sodium periodate (NaIO4) to a final concentration of 0.01 M (i.e. 2.13 mg NaIO4/ml of A + B mixture). Add the glutaraldehyde just prior to use.
Note: The reaction begins as soon as you add glutaraldehyde. (The fixative mixture changes color to yellow upon addition of glutaraldehyde). Use this fixative within minutes of preparation.
Final concentrations: 2% paraformaldehyde, 0.01 M Sodium meta periodate, 0.075 M lysine, and 0.075 M phosphate buffer. Glutaraldehyde concentration should be titrated for each antigen (0-2%).
General Fixative-Making Tips
Stock concentration of ordered fixative: 16% PFA and 8% glut
To make 2% paraformaldehyde with 0.15% glutaraldehyde in 0.1 M PB, prepare 4 % PFA and 0.3% Glut in distilled water. Add equal quantity of 0.2 M PB to make the final concentration of 2% PFA with 0.15% glut in 0.1M PB
To make 24 mls of fixative, add 3ml of 16% PFA and 0.45 ml of 8% glut in 8.55 ml of DW (total volume = 12ml). Add 12 ml of 0.2M PB
Note: If you plan on using any other physiological buffer like 1X PIPES, HBSS or HEPES prepare the final fixative directly in the buffer of choice, e.g., 3 ml of 16 % PFA and 0.45 ml of 8% Glut in 20.55 ml of 1X buffer will give you 24 mls of 2% PFA with 0.15 % Glut in 1X buffer.
Fluorescent labeling of transferrin receptor (green) shows association with chlamydiae (red) in HeLa cells. The higher resolution of electron microscopy confirms that early recycling endosomal markers such as transferrin are recruited, surround, but are not incorporated into the parasitophorous vacuolar membrane.
Nanogold Labeling - TEM
Nanogold, a 1.4 nm gold particle able to penetrate lightly permeabilized cells, can be used to label cellular organelles and external bacterial antigens in a protocol based on the immunoperoxidase procedure. After labeling, the gold size can be amplified for visualization by either silver or gold enhancement.
Nanogold Label for Electron Microscopy
- Hela 2 x 105/ml or other cells on thermanox coverslips in 24 well plates - Infect as appropriate
- Rinse 2X w/ HBSS
- Fix w/ 2-4% paraformaldehyde +0.01- 0.25% glutaraldehyde (titrate glutaraldehyde for each antigen/antibody - if necessary)
2 h @ RT (0 - 2%)
- Rinse 2X w/ PBS (50 mM NaPO4/150 mM NaCl; pH 7.4)
- Incubate specimen for 5 min in 50 mM glycine in PBS (pH 7.4)
- Permeabilize in PBS + 0.01% saponin (titrate saponin; range = 0.005 - 0.05) (Make fresh daily)
5 min @ RT
Block with 1% BSA/PBS for 10 minutes
- Add primary antibody 1:100 in 1%BSA/PBS + 0.01% saponin
1 h @ RT
- Rinse 2X 5 minutes w/ 1% BSA/PBS
- Add secondary antibody: 1:20 nangold (from Nanoprobes, Inc) in 1% BSA/PBS + 0.01% saponin
15 min-1 h @ RT
- Rinse 3X w/ PBS
- Fix with 2.5% glutaraldehyde in 0.1 M PB for 1 hr at room temperature
Gold enhancement step
- Wash coverslips 3x5 minutes with buffer and then 5x5 minutes with diH2O
- Apply enhancement mixture for 15-30 minutes per manufacturer’s instructions. Although we tend to use nanoprobes gold, Aurion’s R-Gent silver enhancement kit is another alternative.
- Wash 5 x 5 minutes with diH2O
- Post fix with 1% osmium tetroxide in dH2O
- Embed as usual (we generally use ethanol for dehydration steps followed by Spurr’s resin)
Colloidal Gold Labeling of External Antigens – TEM/SEM
Surface exposed antigens can be labeled without permeabilizing prior to fixation and preparation for viewing by either TEM or SEM.
Surface antigen labeling of spirochetes prepared by negative staining (A) or thin sectioning (B) viewed by TEM, or by backscatter detection (C) viewed by SEM
Colloidal Gold Labeling of Resin Embedded Samples
Colloidal Gold Labeling of Cryo-Preserved Samples
See cryo-electron microscopy.