TABLE 2.

Assays and their limitations for OS, antioxidant or free radical scavenger expression or activity in the LG, tear film or the ocular surface.

Reference	Goal	Method	Detected Product	Specificity	Limitations
Morrison and Allen (1966)	Identify LPO in LG	Rabbit immune serum against LPO and electrophoresis	Immunoprecipitation in electrophoretic gel	Low	No quantification or cell localization
(Herzog and Miller 1972b)	Determine the location of endogenous peroxidase in acinar cells and fluid	Biochemistry and electron microscopy	Dark spots on electron microscopy and colorimetric assay with guaiacol and pyrogallol test	Low	Limited distinction among peroxidases and catalase activity
Herzog and Fahimi (1976)	Distinguish peroxidase and catalase in acinar cells	Optimum pH evaluation, glutaraldehyde activity to selectively inhibit catalase or peroxidase and electron microscopy	Peroxidase activity and dark spots in electron microscopy	Moderate	Limited distinction from other peroxidase enzymes
Herzog et al. (1976)	Measure the peroxidase release under pharmacologic modulation from the LG cells	Histological and biochemical response to α-adrenergic and muscarinic cholinergic stimulus	Location of dark spots in the acinar cells in vitro and measurement thereof in the tissue and in the supernatant by DAB colorimetric assay	Moderate	Limited distinction among peroxidases isoforms
De (1992)	Measure tissue peroxidase activity and confirm tissue location	Peroxidase purification from tissues and rabbit antiserum anti peroxidase production for western blotting	Comparative enzymatic tissue assays and tissue presence confirmation	Moderate	Potential cross-reaction on western blotting analysis and enzymatic assays
Rios et al. (2005)	Compare LG peroxidase activity as biomarker of DE	Colorimetric assay to obtain peroxidase activity in LG tissues	Colorimetric assay of peroxidase activity	Moderate	Limited distinction among peroxidases isoforms
Jee et al. (2014)	Measure antioxidant enzymes in the tear film	Luminex: magnetic bead-based immunoassay	Catalase and SOD in tear secretion	High	Amount of tear sample and external contamination
Sakai et al. (2015)	Observe the conjunctiva cell culture in the absence of specific antioxidant enzymes	Knockdown of antioxidant enzymes with small interfering RNA	Lipid oxidation and ROS measured by LDH method	High	Immortalized cells in culture have limited compensatory mechanisms
Soria et al. (2017)	Comparative expression of peroxidase and other tear film contents in different diseases	Liquid chromatography with tandem mass spectrometry and quantitative analysis with spectral APEX proteomics	Identification and quantification of enzymatic Redox markers in the tears	High	Tear volume availability and collection method

DAB: 3-3 diaminobenzidine tetrahydrochloride; LDH: lactate dehydrogenase.