SPI-Mark™ Colloidal Gold Reagents
Specific Immunolabeling Protocols
Immunolabeling following In-Situ hydridization for LM
Methods for performing In-Situ hydridization of nucleic acids in embedded EM
tissue sections are widely reported in the literature and no attempt is made
here to repeat them. The steps for In-Situ hydridization are: specimen
preparation; probe labeling; pre-hydridization; hydridization with a specific
labelled probe (labelled with biotin or digoxigenin), followed by antibody
incubations. If biotin labelled probes have been employed then Goat anti-biotin
or streptavidin gold conjugates are used for detection. If digoxigenin labelled
probes are used then Sheep anti-digoxigenin gold conjugates are employed.
(A) Perform the In-Situ hydridization steps according to a suitable protocol and
finish with stringency washing procedures to remove unbound probe from the
section.
(B) Place on the section a 50µl droplet of 10% heat inactivated normal serum
(of gold labelled animal species, e.g. for Goat anti-biotin: gold use normal goat
serum; for Sheep anti-digoxigenin: gold use normal sheep serum; for
streptavidin use nothing) in TBS or PBS buffer and incubate for 10 min.
(C) Replace with a 50µl droplet of gold conjugate diluted in buffer A. Incubate
1-4 hours.
(D) Wash the slide thoroughly with distilled water for 30 min to remove
unbound gold conjugate.
(E) Fix with 1% glutaraldehyde in water for 10 min to bind the gold conjugate.
Wash thoroughly in distilled water for 10 min while agitating to remove
aldehyde groups.
(F) Place on the slide a 50µl drop of freshly mixed
silver enhancing solution
for a period of 5-10 minutes. Determine the time
experimentally while observing under the LM. Avoid the use of excessive light
intensity for prolonged periods.
(G) Wash thoroughly in distilled water to stop the reaction.
(H) Counterstain with normal histological stains.
(I) Mount the section in BIOMOUNT™ to avoid the fading of the silver enhancement.
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Thursday May 17, 2012
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