Publication protocol
After resection, all the collected tissues were placed into 10% neutralized formalin solution (HT50-1-1, Sigma-Aldrich, Co), and then they were processed with a graded series of alcohols and xylene, and finally they were incubated into paraffin waxes. Four-micrometer-thick sections were cut from these paraffin-embedded tissues, deparaffinized using xylene and rehydrated using a graded series of alcohols. Antigen retrieval was performed using a citrate buffer, pH 6.0, at 90°C for 2 cycles of 15 min each. Endogenous peroxidase activity was blocked by the incubation with 3% hydrogen peroxide for 5 min. A serum-free protein block (Dako, Glostrup, Denmark) was applied for 30 min, and the slides were incubated overnight at 4°C with the following antibodies: rabbit polyclonal against GS (clone ab 73593; Abcam Co, UK) diluted 1:1,000, chicken polyclonal against GFAP (clone ab 4674; Abcam) diluted 1:1,000, and rabbit polyclonal against caspase-3 (clone ab 2302; Abcam) diluted 1:10. A 2-step technique was used for the visualization of the antibodies (Envision K 5007; Dako, Glostrup, Denmark), and diaminobenzidine solution was used as chromogen. The slides were then counterstained with hematoxylin for 5 min and were coverslipped with an aqueous-based mounting medium. Matching tissues were used as positive controls for each antibody and omission of the primary antibody as a negative control. Eosin-hematoxylin slides were digitized with a digital camera (Nikon DS-2MW; Nikon Corp, Tokyo) attached to a light microscope (Nikon eclipse 80i; Nikon Corp, Tokyo) under ×200 magnification, and the images were stored as high-quality compressed files in jpg format. Image analysis was performed using the Image Pro Plus 5.1 software (Media Cybernetics, MD, USA). Brown diaminobenzidine staining was indicative of immunohistochemical expression and was considered positive, whereas the blue hematoxylin counterstain was considered negative, as previously described. Briefly, 2 parameters were used for each slide: the average intensity levels of brown staining (measured using arbitrary units on a linear scale from 0 [representing black] to 255 [representing white]) and the average percentage of the extent of brown staining. The average values for each slide were calculated as the mean intensity levels of brown staining and the mean percentage of the extent of brown staining [15].
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