SPI-Mark™ Colloidal Gold Reagents
Trouble shooting (microscopy)
Poor labeling
(A) Antigen absent or destroyed by preparative procedures. Use a different
procedure (e.g. fixes, cryosections, resins). Check that the temperature of the
tissue does rise too high during embedding or polymerisation.
This might be the time to consider the
SPI-Chem™ Low Acid GMA Embedding Kit.
(B) Antigen present in genuinely low amounts. Use longer incubation times
more concentrated primary antibody.
(C) Antibody poor due to low titre, age, poor storage, wrong dilution, excessive
freezing and thawing, low avidity, wrong antibody, no antibody. Change and
check the antibody. Check the positive controls.
(D) Primary antibody too concentrated, producing the hook effect. Use greater
dilutions (1/100-1/10,000).
(E) Wrong gold conjugate. Check and repeat the incubation.
(F) pH of buffer is wrong (excessive alkali or acid). Check solutions.
(G) Insufficient salt content in buffer to allow antibody interactions. Salt content
should be at least 50mM.
(H) Section not exposed to solutions (i.e. wrong way up) if on a plastic film.
(I) labeling not visible due to heavy stains (e.g. for 5nm particles). Reduce
counterstaining. Use higher magnification (e.g. 200,000x for 5nm, 100,000x for
10nm, 80,000x for 15nm, 50,000x for 20nm).
(J) Silver enhancement procedure is incomplete (stain is light brown or absent).
Use longer enhancement times. Check positive controls.
(K) Counterstaining is masking the silver stain. Use less counterstain.
(L) Silver stain fades after some time. Remove coverslip with xylene and wash in
developer. Remount in BIOMOUNT™.
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Thursday May 17, 2012
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