Aquatex fluid

The hind paws skin was fixed in 4% neutral buffered formalin and then after 24 h, samples were embedded in paraffin, sectioned into 4–5 μm thick sections, and stained with hematoxylin and eosin (H&E) to analyze the general histopathological changes. Immunoreactivities of inducible nitric oxide synthase (iNOS) and NF-kB profiles were investigated using purified primary antibodies with avidin-biotin-peroxidase (ABC) and peroxidase substrate (Pierce™ Peroxidase IHC Detection Kit, Thermo Fisher Scientific, Waltham, CA, USA). Briefly, the endogenous peroxidase activity was blocked by 0.3% H₂O₂ for 30 min in a humidity room after heating (95–100 °C) the sample. The sections were then incubated with primary antibody overnight at 4 °C in a humidified room followed by incubation with biotinylated rabbit anti-mouse secondary antibody (Dako system kit, Santa Clara, CA, USA) and avidin-biotin complex (ABC) reagents for 1 h at 30 °C in a humidified room. Finally, the specimens were counterstained with hematoxylin, dehydrated, and mounted using Aquatex fluid (Merck KGaA, Darmstadt, Germany).

The immunolocalization technique used for TIMP-2 and Bax was performed on 3 to 4 μm thick sections according to Pedrycz and Czerny [30 (link)]. For negative controls, the primary antibody was omitted. In brief, mouse anti-Bax or anti-TIMP-2 (diluted1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies were incubated with sections for 60 min, diluted in TBS (Tris-buffered saline)/1 % BSA (bovine serum albumin). A biotinylated secondary antibody directed against mouse immunoglobulin (Biotinylated Link Universal–DakoCytomation kit, supplied ready to use) was then added and incubated for 15 min, followed by horse radish peroxidase conjugated with streptavidin (DakoCytomation kit, supplied ready to use) for an additional 15 min of incubation. 3-amino-9-ethylcarbasole (AEC) (DakoCytomation kit, supplied ready to use) was then applied for 15 min, yielding a reddish brown color at the sites of immunolocalization of the primary antibodies. Specimens were counterstained with hematoxylin for 1 min and mounted using Aquatex fluid (Merck KGaA, Germany). The staining intensity was graded as very weak, weak, medium, or strong. All sections were incubated under the same conditions and at the same time with the same concentration of antibodies to ensure that the immunostaining would be comparable among the different experimental groups.