Tissuefaxs workstation

Formalin-fixed renal tissue sections were stained for CD45 using immunohistochemistry. Sections (4-µm-thick) were deparaffinized with xylene, rehydrated in a graded alcohol series, and then transferred to citrate buffer solution (pH 6.0). Slides were placed in a pressure cooker and heated by microwaving for 10 min to enhance antigen retrieval. After cooling, the kidney sections were immersed in a hydrogen peroxide solution (DAKO, Carpinteria, CA) for 30 min to block endogenous peroxidase activity, followed by overnight incubation at 4°C with serum-free protein block (DAKO). The next day, the slides were incubated with a 1:100 dilution of anti-mouse CD45 monoclonal antibody (BD Biosciences, San Jose, CA) for 1 h at room temperature. After being rinsed, the CD45-stained sections were incubated for 30 min at room temperature with a secondary antibody using a Dako REAL EnVison kit (DAKO). Subsequently, 3,3’-diaminobenzidine tetrahydrochloride (DAKO) was applied to the slides to produce a brown color and then the slides were counterstained with Mayer’s hematoxylin solution (DAKO).
To calculate the percentage of CD45-positive cells in kidney samples, whole fields of slides including both cortex and medulla were scanned and analyzed with a TissueFAXS work station (Tissue Gnostics, Vienna, Austria), as described previously (17 (link)).

Formalin-fixed renal tissue sections were immunostained for detection of CD45 as follows. Sections (4-μm-thick) were deparaffinized with xylene, rehydrated in a graded alcohol series, and placed in a citrate buffer solution (pH 6.0). Slides were placed in a pressure cooker and heated for 10 min to enhance antigen retrieval. After cooling, the kidney sections were immersed in a hydrogen peroxide solution (Dako, Carpinteria, CA) for 30 min to block endogenous peroxidase activity, followed by overnight incubation at 4°C with serum-free protein block (Dako). The next day, the slides were incubated with a 1:100 dilution of anti-mouse CD45 monoclonal antibody (BD Biosciences, San Jose, CA) for 1 h at room temperature. After being rinsed, the CD45-stained sections were incubated for 30 min at room temperature with a secondary antibody using a Dako REAL EnVison kit (Dako). Subsequently, 3,3′-diaminobenzidine tetrahydrochloride (Dako) was applied to the slides to produce a brown color, and the slides were counterstained with Mayer's hematoxylin solution (Dako).
A TissueFAXS workstation (Tissue Gnostics, Vienna, Austria) was used to analyze and calculate the percentage of CD45-positive cells in kidney samples, as described previously (18 (link)).

Immunohistochemical staining of a cluster of differentiation (CD) 45 (leukocyte common antigen) using formalin-fixed renal tissue sections was performed with reference to a previous report^{30 (link)}. The renal tissue sections (4-µm-thick) were deparaffinized using xylene, rehydrated in a graded alcohol series, and then transferred to citrate buffer solution (pH 6.0). The slides were placed in a pressure cooker and heated in microwave for 10 min to enhance antigen retrieval. After cooling, the slides were immersed in hydrogen peroxide solution (Dako, Carpinteria, CA) for 30 min to block the endogenous peroxidase activity. After overnight incubation at 4 °C with serum-free protein block (Dako), the slides were incubated at room temperature for 1 h with a 1:100 dilution of monoclonal rat anti-mouse antibody to CD45 (BD Biosciences, San Jose, CA). After being rinsed, the CD45-stained sections were incubated for 30 min with a secondary antibody using a Dako REAL EnVison kit (Dako) at room temperature. Staining with 3,3′-diaminobenzidine tetrahydrochloride (Dako) was performed on the slides to extract brown color, then counterstaining with Mayer's hematoxylin (Dako) was performed. A TissueFAXS workstation (Tissue Gnostics, Vienna, Austria) was used to analyze and calculate the percentages of CD45-positive cells infiltrated into renal tissues, as described previously^{30 (link)}.