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H.A.854, E.O.D. N-056657, SITRO, 2°50h 06′ ± 10′ The concentration = 0.09 mg OAA, 1.25×10^−2^ M; pH 8; 12 h: 3 µl; 72 h 15 min: 1.7 µl; 120 min 36.07 µl; 144 h 26 µl; 240 h 71 µl; 152 µl 3 µl 7-dihydroxyphenylalanine and hexakisornithine.
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The solution was partitioned into 2.0 µl of CH~3~OH with a heparin-*rich* heparin layer and incubated overnight in the dark. Incubation was then continued to the last four hours of incubation. 2.2. Test for Anti-NMDA Signals {#sec2dot2-molecules-21-00329} ——————————- Comparable amounts of NMDA antagonist (clupidumab, etanercept, or golimumab) were used as negative controls as previously reported, respectively. Briefly, the samples were administrated to the indicated mice by intravenous injection, the mice were randomly distributed into six four-group groups consisting of 5 mice per group (*n* = 6 in each hand) \[[@B54-molecules-21-00329]\], and they underwent treatment with 1 µg of clupidumab, golimumab, or 5 µg of each vehicle. Each animal was tested once per mouse at 60 d after intraperitoneal injection. Salivary glands (adults)’ skin and eyes (adults), mammary gland, and testis (adults) were removed immediately after injection. Thereafter, at the end of the experiment the tissues were removed and divided into three groups (4 groups were used in the pilot study) – control, clupidumab, golimumab, or 5 µg saline solution; each group was analyzed by two independent tests and mice were sacrificed in groups.
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2.3. Immunohistochemical Examination {#sec2dot3-molecules-21-00329} ————————————- Mice were euthanized with chloral hydrate, serrated in 50% acetone, and fixed with 4% formaldehyde for one hour at room temperature. The fixed tissues were embedded in OCT (Veidom, Piscataway, NJ, USA). Decalorized tissue sections were sectioned 4 µm using a MIVO-II (Leica, Munich, Germany). The sections were dried at 40o F before being stained with gold and mounted under x-irradiance for air interlameous (A) and OHC. The sections were analyzed using an ATCO (Nederlandse zondereis): 45 nm, 65 nm, 45 nm, and 40 nm. The sections were analysed using the Zia-I (Vishagard, Orsay, Switzerland). Immunoreactivity against NMDA was semi-quantified with fluorocals Alexa Fluor 405 and 558. 2.
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4. Real-Time Quantitative PCR and Statistical Analysis {#sec2dot4-molecules-21-00329} —————————————————— RNA Home were prepared for RT-qPCR analysis using an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Each condition was statistically analyzed 2 independent experiments (two-way ANOVA). Data are expressed as mean ± SD for one of the six mouse groups of 6 animals of each experimental group. Results were processed as described previously with no a multiple-counts correction; the fold change and statistical significance (p-value threshold: 0.025) were discussed relative to the mean control value in the normal region \[[@B55-molecules-21-00329]\]. 2.5. Statistical Analysis {#sec2dot5-molecules-21-00329} ————————- Statistical significance was assessed by one-way ANOVA and the probability p-value was determined using the Bonferroni-Holm method. Data were analyzed in SPSS 20 software package.
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