We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

In vivo total antioxidant capacity: comparison of different analytical methods.

Three assays were compared for the determination of total antioxidant capacity in human serum: the oxygen radical absorbance capacity (ORAC) assay, the Randox Trolox-equivalent antioxidant capacity (Randox-TEAC) assay, and the ferric reducing ability (FRAP) assay. There was a weak but significant linear correlation between serum ORAC and serum FRAP. There was no correlation either between serum ORAC and serum TEAC or between serum FRAP and serum TEAC. The effect of dilution on the serum TEAC value and the use of inhibition percentage at a fixed time, without considering the length of inhibition time in the quantitation of results, adversely affected the Randox-TEAC assay. The FRAP assay is simple and inexpensive but does not measure the SH-group-containing antioxidants. The ORAC assay has high specificity and responds to numerous antioxidants. By utilizing different extraction techniques in the ORAC assay, one can remove serum proteins and also make some gross differentiation between aqueous and lipid-soluble antioxidants. However, the ORAC assay requires ∼60 min more than the FRAP or Randox-TEAC assay to quantitate results.

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Comparison of different analytical methods for assessing total antioxidant capacity of human serum

The antioxidant activities of methanol and ethyl ether extracts obtained from Thymus zygis, collected during the flowering or non-flowering period, were evaluated and compared. To investigate this potential, extracts were tested on their capacity to react with diphenylpicrylhydrazyl (DPPH) in a homogeneous medium, and to inhibit Fe2+/ascorbate-induced membrane lipid peroxidation, as estimated by the formation of thiobarbituric acid-reactive substances (TBARS). Although methanol extracts reduce DPPH radicals more efficiently than ethyl ether extracts, suggesting a potent radical scavenger activity, the ethyl ether extracts were found to be most active in inhibiting lipid peroxidation in sarcoplasmic reticulum (SR) membranes. In addition, both extracts present peroxyl and superoxide radical scavenging activities. Peroxyl radicals were generated by the water soluble 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) azoinitiator, and the scavenging activities of the extracts were measured by the inhibition of cis-parinaric acid (PnA) fluorescence decay in SR. Superoxide radicals were generated either by an enzymatic or a non-enzymatic system, and the scavenger ability was evaluated by the inhibition of nitroblue tetrazolium reduction. Methanolic extracts are more potent as scavengers of peroxyl and superoxide radicals than the ethyl ether extracts. Apparently, there is a relationship between antioxidant potency and the total phenolic groups content in each extract.

Antioxidant activities of some extracts of Thymus zygis

Review on some plants of Indian traditional medicine with antioxidant activity.

In this study, we examined the effect of the Maharishi Ayur-Veda herbal mixtures (MAHMs) Maharishi Amrit Kalash-4 and -5 (M-4 and M-5), MA-631, and Maharishi Coffee Substitute (MCS) on low-density lipoprotein (LDL) oxidation and compared the potency of these mixtures to ascorbic acid, α-tocopherol, and probucol. LDL was incubated in 95% air and 5% CO2, with or without 3 μM Cu+2, in the presence or absence of MAHMs, for 6 or 24 h. In a separate experiment, LDL was incubated as above except MAHMs were added at 0, 1.5, and 3.5 h after incubation started to assess their effect on initiation and propagation of LDL oxidation. Our results demonstrate that MAHMs caused concentration-dependent inhibition of LDL oxidation as assessed by thiobarbituric acid-reactive substances and electrophoretic mobility. The MAHM showed more antioxidant potency in preventing LDL oxidation than ascorbic acid, α-tocopherol, or probucol. Also, MAHMs inhibited both initiation and propagation of cupric ion-catalyzed LDL oxidation. These results suggest the importance of further research on these herbal mixtures in the investigation of atherosclerosis and free radical-induced injury.

Inhibition of human low-density lipoprotein oxidation in vitro by Maharishi Ayur-Veda herbal mixtures.

Effect of herbal mixture student Rasayana on lipoxygenase activity and lipid peroxidation.

We have compared the avidin-biotin complex immunoperoxidase method with a modified horseradish peroxidase a vidin D technique. Use of the horseradish peroxidase avidin D procedure saved time without compromising quality of the staining, by reducing the amount of time for methanol-hydrogen peroxide block, serum block, antibody incubations, horseradish peroxidase avidin D incubation, and Gill-3 hematoxylin counterstain, and cut costs by using higher dilutions of primary antibodies. Other helpful modifications include the utilization of phosphate-buffered saline, batching slides for secondary antibody and horseradish peroxidase avidin D, and instituting a standard development time.

An Improved Immunoperoxidase Technique Using Horseradish Peroxidase Avidin D

Excess free radicals are linked to many diseases, including aging, atherosclerosis, and cancer. Previously, we have shown that MA-631 (a complex herbal mixture) inhibits human low-density lipoprotein (LDL) oxidation and may play a role in prevention of atherosclerosis. In this study we further evaluated the in vivo and in vitro antioxidant activity of MA-631. Both the alcoholic and aqueous extracts of MA-631 inhibited enzymatic- and nonenzymatic-induced rat liver microsomal lipid peroxidation in a concentration-dependent manner. The thiobarbituric acid-reactive substances (TBARS) values (nmol malondialdehyde (MDA)/mg microsomal protein) were 1.43 ± 0.18 for microsomes alone (baseline for enzymatic system), 19.63 ± 2.50 for microsomes + reduced nicotinamide adenine dinucleotide phosphate (NADPH) (oxidation without inhibitor), 9.89 ± 1.41 for heated microsomes (baseline for nonenzymatic system), and 27.15 ± 0.08 for microsomes + ascorbate (oxidation without inhibitor). The concentrations (μg/2 ml) of MA-631 which produced 50% inhibition (IC 50 ) of enzymatic- and non-enzymatic-induced lipid peroxidation were 15.2 ± 2.0 and 17.0 ± 2.6, respectively for the aqueous extract, and 4.3 ± 0.8 and 6.4 ± 1.2, respectively, for the alcoholic extract. A 2% MA-631 (w : w) supplemented diet fed to rats for three weeks inhibited in vivo, toluene-induced microsomal lipid peroxidation in the brain, kidney, liver, and heart. These results imply that MA-631 may be useful in the prevention of free radical-linked diseases.

In vitro and in vivo inhibition of microsomal lipid peroxidation by MA-631.

For paraffin sections to be stained by the immunoperoxidase method, we compared the conventional slide drying technique with a microwave technique. The conventional technique consists of drying the slides for 1 hour in a 58°C oven. The microwave technique consists of drying the slides for 1 minute in a General Electric Spacemaker II microwave oven. In addition to time savings, antigen staining was enhanced by the microwave drying in the majority of antibodies tested.

Ellen M. Kauffman

Papers

Inhibition of human low-density lipoprotein oxidation in vitro by Maharishi Ayur-Veda herbal mixtures.

Effect of herbal mixture student Rasayana on lipoxygenase activity and lipid peroxidation.

An Improved Immunoperoxidase Technique Using Horseradish Peroxidase Avidin D

In vitro and in vivo inhibition of microsomal lipid peroxidation by MA-631.

Improved Immunoperoxidase Staining Using Microwave Slide Drying