Immunogold labeling for two or more antigens

This is the favorite electron microscopic immunolabeling protocol in the laboratory of Dr. G. V Childs at the University of Texas Medical Branch . The purpose of this protocol is to allow labeling for two (or more) antigens with sensitive, small protein-A conjugated immunogold markers . This protocol will illustrate our technique for labeling luteinizing hormone (LH), follicle stimulating hormone antigens (FSH) or GH antigens in pituitary cells. In the Figure, the large gold marks sites of FSH antigens in the smaller storage granules. The small gold marks the site of GH antigens scattered in the largest storage granule on the left. This cell is storing both GH and FSH antigens. For more information about the multipotential somatogonadotrope, see the web page on Growth hormone and gonadotropin co-expression just before ovulation.


First, make Protein A gold--antibody complex (1:50) stock solutions. We obtain all colloidal gold products from Goldmark Biologicals

For LH antigens: 5 microliters Anti-LH (1:2) + 120 microliters of 5 nm gold conjugate

For FSH antigens: 50 microliters Anti-FSH (1:25) + 50 microliters of 10 nm gold conjugate

For a third antigen, use 15 nm gold conjugate with a third antibody.

Mix & incubate at 37 degrees for 2 hours to form the labeling complex

Make the solution that "etches the embedding plastic---dissolve 50 mgs. sodium metaperiodate in 10 ml. millipore filtered water

Make the blocking solution-- 5% BSA in 0.025M TRIS buffer; use for blocking & as diluent buffer
Make wash buffer-- 0.025M TRIS buffer ph 8.2
Prepare 1% glutaraldehyde in millipore filtered water


Cut ultrathin sections and place on nickel grids. Etch sections on one side with 0.5% sodium metaperiodate---10 mins. room temperature.
Immerse/jet-wash 20 times in 0.025M TRIS buffer; blot lightly.
Block with 5% Bovine serum albumin in 0.025M TRIS buffer---15 mins. room temperature; blot
Incubate grids in diluted antibody-gold complex (30 microliters/drop) for 2 hours at 33-34 degrees using special tissue culture plates (e.g. 1:3,000 protein A gold-Anti-LH/0.025M TRIS buffer + 5% bovine serum albumin).
Immerse/wash in 0.025m TRIS buffer & blot against filter paper to dry
Invert grid to label the other side of the ultrathin sections with the second antibody, etch   other side with 0.5% NaIO4---3 mins. room temperature,
Immerse/wash in 0.025M TRIS buffer; blot
Block with 5% BSA in 0.025M TRIS buffer---15 mins. room temperature, blot
Incubate in the diluted second antibody-gold complex for 2 hours at 33-34 degrees (1:1,500 pAG10-Anti-FSH/0.025M TRIS buffer + 5% BSA)
Immerse/wash the grids in 0.025M TRIS buffer & dry by blotting on filter paper
A third antigen can be detected on the first side by 15 nm protein A antibody gold complexes as described for the first, however, the etching step can be omitted. Usually we detect only two antigens at a time.
Fix the grids in 1% glutaraldehyde---10 mins. room temperature
Immerse/wash in millipore filtered water & dry by blotting on filter paper
Counterstain with uranyl acetate & lead citrate
Make solutions one day before use:
lead citrate---dissolve 10-40 mg. in 10 ml. Millipore filtered water, add 100 microliters of 10N NaOH, mix solution vigorously
uranyl acetate---dissolve 0.8 gm. in 10 ml/ absolute ethanol, mix vigorously; solution must be saturated
immerse grid in uranyl acetate drops---5-7 mins. room temperature
wash in 25% ethanol---1X by immersion
wash in Millipore filtered water---2X by immersion
dry grids on filter paper---10 mins. room temperature
float on lead citrate drops---5 mins. room temperature
0.02N sodium hydroxide---2X by immersion, alternating with millipore filtered water---2X by immersion 
also dry grids on filter paper

NOTE: lead citrate combines readily with CO2, so keep the dish covered & don't breathe on it. Sodium hydroxide pellets may be used to take up CO2 from air. Make a chamber with sodium hydroxide pellets in one side and a teflon or wax surface for staining the grids on the other side.

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Other Protocols:
Affinity Cytochemistry
In situ hybridization histochemistry

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