Immunohistochemical Analysis of Cellular Stress Markers

After de-paraffinization of sections, the slides were autoclaved in 0.1M sodium citrate pH 6 for antigen retrieval step (30 (link)). The slides were allowed to cool and washed with PBS twice times. Slides were incubated with 5% normal serum depending upon the source of secondary antibody used. The slides were incubated with primary antibodies at 4°C overnight (HSP70, 8-oxoguanine p-JNK, COX2, GFAP) from Santa Cruz Biotechnology at 1:100 dilution. Next morning, after washing with PBS, fluorescent labeled secondary antibodies (Santa Cruz Biotechnology) as 1:50 dilution were used for signal amplification in dark chamber, followed by mounted with UltraCruz mounting medium (Santa Cruz Biotechnology). The slides were pictured with confocal scanning microscopes (Flouview FV 1000, Olympus, Japan) and fluorescence intensity was quantitatively analyzed by ImageJ and expressed as the relative integrated density of the samples relative to the sham.

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Shah F.A., Li T., Kury L.T., Zeb A., Khatoon S., Liu G., Yang X., Liu F., Yao H., Khan A.U., Koh P.O., Jiang Y, & Li S. (2019). Pathological Comparisons of the Hippocampal Changes in the Transient and Permanent Middle Cerebral Artery Occlusion Rat Models. Frontiers in Neurology, 10, 1178.

Publication 2019

HSP70 COX2 GFAP fluorescent labeled secondary antibodies UltraCruz mounting medium Flouview FV 1000

8 oxoguanine Antibodies Antibody Antigen Confocal scanning microscopes Cox2 Dilution Fluorescence Fluorescent antibodies Gfap Hsp70 Serum Sodium citrate

Corresponding Organization : Second Hospital of Shandong University

Other organizations : Riphah International University, Xi'an Jiaotong University, Zayed University, Khyber Medical College, Khyber Medical University, Nantong University, Huazhong University of Science and Technology, Shenzhen Center for Disease Control and Prevention, Gyeongsang National University

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Variable analysis

independent variables

Antigen retrieval method (autoclaving in 0.1M sodium citrate, pH 6)

dependent variables

Fluorescence intensity of HSP70, 8-oxoguanine, p-JNK, COX2, GFAP

control variables

Concentration of primary antibodies (1:100 dilution)
Concentration of secondary antibodies (1:50 dilution)
Incubation time for primary antibodies (overnight at 4°C)
Mounting medium (UltraCruz)

controls

Sham control (used as reference for relative fluorescence intensity)

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