SPI-Mark™ Colloidal Gold Reagents
Specific Immunolabeling Protocols
Pre-Embedding labeling Tissues for EM
(A) Dissect the tissue to the smallest possible size (e.g. 0.5mm) for handling and
good penetration.
(B) Lightly fix tissue in
2-4% paraformaldehyde
+
0.1% glutaraldehyde
for 30-60 min depending on tissue size.
(C) Wash tissue very thoroughly in
PBS
(e.g. 30-60 min) to remove excess aldehyde groups.
(D) Optionally permeabilise tissue with 0.1% Tween 20 or
Triton®-X 100 nonionic surfactant for 15-30 min.
(E) Wash tissue very thoroughly in
Buffer A for 1 hour.
(F) Incubate tissue with 20 x volume of primary antibody, diluted in Buffer A, for 1-4 hours while agitating.
(G) Wash in buffer A very thoroughly, i.e. for 1 hour while agitating, to remove excess primary antibody.
(H) Incubate tissue with gold conjugate suitably diluted in Buffer A for 1-4 hours
while agitating. For most applications only 1nm gold conjugates will penetrate
the cell membranes. 1nm gold conjugates are usually diluted to 1/100-1/400 incubations.
(I) Wash tissue very thoroughly in Buffer A (30 min) and then in PBS for 30 min
while agitating to remove excess gold conjugate.
(J) Fix tissue in 1% glutaraldehyde for 10 min to strengthen antibody binding to tissue.
(K) Wash thoroughly in water for 30 min while agitating to remove excess aldehyde and PBS.
Optional silver enhancing:
(L) Immerse the tissue in fresh
silver enhancing solution for 5-15
min. Experiment with a number of tissue blocks to determine the time required.
(M) Wash thoroughly in distilled water to stop enhancing.
(N) Continue through to dehydration and resin embedding for EM or investigations.
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Thursday May 17, 2012
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