SPI-Mark™; Colloidal Gold Reagents
Trouble shooting (microscopy)
Excessive Background Labeling
(A) Ionic concentration too low in solutions. Use increased salt concentration (up to 2.5%).
Add BSA or normal goat serum (not for Protein A, G, A/G) to approximately 1% in incubation solutions.
(B) Inadequate washing between incubations. Thoroughly wash (hours if
necessary).
(C) Non-specific charge attraction of antibody. Raise the pH of the buffer to
pH8.2 - 8.5 to reduce the positive charge on tissue proteins.
(D) Hydrophobic attraction of gold particles to tissue components. Increase the
Tween 20 content to 1 - 2%.
(E) Non specific attraction of second antibody to tissue components. Include
higher levels of normal serum (e.g. normal goat serum for Goat anti-rabbit:
gold) in the buffer, up to 5%.
(F) Impurities in the normal serum used. Omit the serum and observe the
results.
(G) High levels of sulphur attracting gold particles in the tissue or embedding
resin. Change to acrylic resins or include up to 0.01% gelatin in the gold
conjugate for that step only.
(H) Free aldehyde groups in the tissue. Reduce by floating sections on 0.5M
ammonium chloride for 1 hour before incubations.
(I) Primary antibody concentration too high. Dilute by orders of magnitude.
Wherever possible use affinity purified antibodies.
(J) Gold conjugate concentration too high. Dilute further.
(K) Inadequately fixed tissue. Necrotic and damaged cells will also stain nonspecifically.
Improve fixation conditions and use smaller pieces of tissue.
Check the positive control.
(L) Osmium tetroxide fixation may introduce excess charge into tissue. Omit
osmium or wash thoroughly after fixing.
(M) Excessive exposure to silver enhancing solutions, especially for low signal.
Reduce the development time.
(N) Excessive exposure to light during silver enhancement. Reduce the light
intensity.
(O) Spontaneous precipitation of silver due to impurities in glassware and the
water. Use ultraclean glassware and distilled/deionised water of high purity.
(P) Spontaneous precipitation of silver onto endogenous metals in tissue (e.g.
Zn, Fe, Cd, etc). Carefully check the negative controls.
(Q) Non specific attraction of gold to lysine when used as a tissue binding
agent on slides. Avoid the use of lysine and use BIOBOND™; instead.
To Ask a Question or Make a Comment
To Place an Order or Request a Quote
Return to:
Thursday May 17, 2012
© Copyright 2006 - 2012. By Structure Probe, Inc.
Contacting SPI Supplies and Structure Probe, Inc.
All rights reserved.
All trademarks and trade names are the property of their respective owners.
Privacy Policy
Worldwide Distributors, Representatives, and Agents
