Hydrogen-Mediated Activation of the Nrf2/ HO -1 Signaling Pathway Improves Cognitive Impairment in Sleep-Deprived Mice methods
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Nissl Staining
After routine deparaffinization and rehydration, tissue sections were treated with Nissl staining solution for 30-60 s. Excess stain was removed by washing with distilled water, followed by differentiation in 1% glacial acetic acid. Processed sections were coverslipped with buffered glycerol mounting medium and examined by light microscopy.
Enzyme-Linked Immunosorbent Assay ( ELISA )
Brain tissue samples were mechanically homogenized and stored at -20°C for 24 h. After two complete freeze-thaw cycles at 2°C-8°C to disrupt cells, homogenates were centrifuged at 5000 × g for 5 min to collect supernatant. ELISA was conducted following kit instructions. Commercial kits were utilized to detect cytokines: mouse tumor necrosis factor-α (TNF-α; KE10002), interleukin (IL)-1β (KE10003), and IL-6 (KE10007) from Proteintech (USA), as well as oxidative stress markers glutathione peroxidase (GSH-PX, A005-1), malondialdehyde (MDA, A003-1), and superoxide dismutase (SOD, A003-1) from Nanjing Jiancheng Bioengineering Institute (China).
Immunohistochemistry
Following standard deparaffinization and rehydration steps, tissue sections underwent antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) followed by gradual cooling to ambient temperature. After three 3-min washes with phosphate-buffered saline (PBS, 0.01 M), endogenous peroxidase activity was blocked with 1% periodic acid solution (10 min incubation) and subsequent PBS rinses (3 × 3 min). Primary antibody incubation occurred at 4°C for 12-16 h using these antibodies (all from Proteintech): postsynaptic density protein 95 (PSD95, 1:200), synaptophysin (SYN, 1:200), Nrf2 (1:200), and HO-1 (1:200). Secondary detection involved 30-min incubation at 37°C with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG). Color development employed a 3,3′-...
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After routine deparaffinization and rehydration, tissue sections were treated with Nissl staining solution for 30-60 s. Excess stain was removed by washing with distilled water, followed by differentiation in 1% glacial acetic acid. Processed sections were coverslipped with buffered glycerol mounting medium and examined by light microscopy.
Brain tissue samples were mechanically homogenized and stored at -20°C for 24 h. After two complete freeze-thaw cycles at 2°C-8°C to disrupt cells, homogenates were centrifuged at 5000 × g for 5 min to collect supernatant. ELISA was conducted following kit instructions. Commercial kits were utilized to detect cytokines: mouse tumor necrosis factor-α (TNF-α; KE10002), interleukin (IL)-1β (KE10003), and IL-6 (KE10007) from Proteintech (USA), as well as oxidative stress markers glutathione peroxidase (GSH-PX, A005-1), malondialdehyde (MDA, A003-1), and superoxide dismutase (SOD, A003-1) from Nanjing Jiancheng Bioengineering Institute (China).
Following standard deparaffinization and rehydration steps, tissue sections underwent antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) followed by gradual cooling to ambient temperature. After three 3-min washes with phosphate-buffered saline (PBS, 0.01 M), endogenous peroxidase activity was blocked with 1% periodic acid solution (10 min incubation) and subsequent PBS rinses (3 × 3 min). Primary antibody incubation occurred at 4°C for 12-16 h using these antibodies (all from Proteintech): postsynaptic density protein 95 (PSD95, 1:200), synaptophysin (SYN, 1:200), Nrf2 (1:200), and HO-1 (1:200). Secondary detection involved 30-min incubation at 37°C with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG). Color development employed a 3,3′-diaminobenzidine substrate system (ZSGB-BIO, China). Final processing included sequential dehydration in graded ethanols (60-100%, 5 min per concentration), xylene clearing (10 min), and mounting with neutral gum for light microscopic assessment.
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