Kidney segments were fixed in 4% formalin for 48 h, then embedded in paraffin and cut. Kidney sections were deparaffinized, rehydrated and stained with Masson’s trichrome (Bio-Optica, Milano, Italy). Histological analyses were done by two Pathologists (AFT and DB) blinded to study groups. Damaged area quantification of Masson’s trichrome staining was performed as described by Chen et al. using the ImageJ software34 (link).
Immunohistochemistry staining was performed as previously described5 (link). Primary antibodies against F4/80 (BM-8) (diluted 1:30; Santa Cruz biotechnology Inc., Dallas Texas, USA) and Myeloperoxidase (MPO) (diluted 1:50; Abcam, Cambridge, UK), NF-κB p65 (Bio-Rad, CA, USA), IL-1 and IL-6 (GeneTex, CA, USA) were used for the immunohistochemistry staining. For image processing, Topika Analysis software (Topika, Tel Aviv, Israel) was used. Area quantification of immunohistochemical staining was performed using an ImageJ software as described.
Immunohistochemistry staining was performed as previously described5 (link). Primary antibodies against F4/80 (BM-8) (diluted 1:30; Santa Cruz biotechnology Inc., Dallas Texas, USA) and Myeloperoxidase (MPO) (diluted 1:50; Abcam, Cambridge, UK), NF-κB p65 (Bio-Rad, CA, USA), IL-1 and IL-6 (GeneTex, CA, USA) were used for the immunohistochemistry staining. For image processing, Topika Analysis software (Topika, Tel Aviv, Israel) was used. Area quantification of immunohistochemical staining was performed using an ImageJ software as described.
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