Immunofluorescence staining protocol for co-staining of fetuin-A and CD68 in older human autopsy tissue v1 (protocols.io.syfeftn)

Through modification of the protocol by Adams, we developed a biotin amplification procedure for immunofluorescence staining of immediate early gene proteins and also applied biotin amplification for metal enhancement of diaminobenzidine staining in an immunoperoxidase protocol. Commercially available anti-cFos antisera were used to compare conventional "Elite" avidin-biotin complex reactions with biotin amplification reactions (visualized with peroxidase staining or streptavidin-Texas Red fluorescence). Biotin amplification and peroxidase staining (with or without nickel salts) enabled detection of cFos in stimulated neurons with primary antibody concentrations five- to tenfold lower than the conventional procedure. With immunofluorescence staining, at primary antibody concentrations too low to detect cFos with the conventional biotin-streptavidin fluorescence staining protocol, biotin amplification enabled clear cFos fluorescence staining with both antisera. The fluorescence staining exhibited a high signal-to-noise ratio and enabled antibody concentrations four times lower than those used for conventional ABC "Elite" peroxidase procedures. In conclusion, the application of biotin amplification to cFos immunocytochemical localization has the promise of aiding the scientist in detecting these immediate early gene products.

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Inhibition of lactate dehydrogenase isoenzymes by sodium perchlorate evaluated

Clinical Chemistry ◽

10.1093/clinchem/36.11.1964 ◽

1990 ◽

Vol 36 (11) ◽

pp. 1964-1966 ◽

Cited By ~ 2

Author(s):

G T Sanders ◽

E van der Neut ◽

J P van Straalen

Keyword(s):

Enzyme Activity ◽

Lactate Dehydrogenase ◽

Sodium Perchlorate ◽

Reaction Medium ◽

Final Concentration ◽

Selective Determination ◽

Autopsy Tissue ◽

Human Autopsy ◽

Lactate Dehydrogenase Isoenzymes

Abstract We evaluated a method of measuring lactate dehydrogenase isoenzyme 1 (LD-1) selectively (Clin Chem 1987;33:991-2), in which all other LD isoenzymes were inhibited by adding sodium perchlorate to the reaction medium to a final concentration of 0.825 mol/L. In this study we used the different isoenzymes purified from human autopsy tissue and found that the originally published amount of inhibitor (a) increased the original LD-1 activity and (b) did not eliminate all enzyme activity of LD-2 and LD-3. Interference by LD-2 was further demonstrated. Thus we conclude that this method cannot be used for the selective determination of LD-1 because its results do not accurately reflect the original LD-1 activity.

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