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)
Caution: For all fluid exchanges, remove most of the liquid but not all to avoid drying of sample which adversely affects the ultrastructure.
(Titrate glutaraldehyde for each antigen/antibody. If necessary, glutaraldehyde can be omitted, although te use of glutaraldehyde improves ultrastructural preservation)
(Commercial PBS will work if it doesn’t have any additives like azide)
(Make fresh daily from powder) Include this saponin concentration for all antibody steps)
(Choose appropriate host specific HRP conjugated antibody)
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.
Metal-enhanced DAB substrate kit, e.g., Pierce Protein Biology Products Saponin from Quillaja bark/25 grams, e.g., Sigma AldrichAntibody – HRP conjugates, e.g. Jackson Immunoresearch Lab
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.
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%).
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, 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.
Block with 1% BSA/PBS for 10 minutes
Gold enhancement step
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
See cryo-electron microscopy.
Last Updated August 16, 2013
Last Reviewed August 16, 2013