Mayer s hematoxylin solution

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)).

A representative fraction of each tumor and whole lungs from all the animals described were fixed in 4% formalin and then paraffin embedded (FFPE) using standard protocols. For Tomato detection in tumors and lungs, 3 μm slices were stained with Anti-RFP antibody ab124754 (Abcam, Cambridge, UK) diluted 1:400 in PBS containing 0.05% Tween 20. Revealing was performed using Mach 1 HRP-polymer (Biocare Medical, Concord, CA, USA) incubation followed by the revelation with Betazoid DAB (Biocare Medical). Negative controls were conducted by omitting the primary antibody. Nuclei were counterstained with Mayer's hematoxylin solution (Dako, Milan, Italy).
Double immunofluorescence staining was performed on FFPE 3 μm slices of tumors and lungs. We used the Anti-GFP, rabbit IgG fraction, biotin-XX conjugated monoclonal antibody (Life Technologies) diluted 1:200, and the Anti-RFP antibody ab124754 (Abcam) diluted 1:400. Secondary antibodies were Streptavidin Alexa Fluor^® 488-conjugated (1:500) and Alexa Fluor^® 594-conjugated donkey anti rabbit (1:500) respectively (Life Technologies). Nuclei were counterstained with DAPI (10 μg/ml). Sections were mounted with the antifade medium FluorPreserve Reagent (Calbiochem, Merck-Millipore) and analyzed with an Olympus Fluoview FV1000/TIRF laser scanning confocal microscope.

Mayer’s hematoxylin solution was purchased from Dako (Glostrup, Denmark), alcoholic eosin Y solution, trypan blue 0.4 % solution and DAPI were purchased from Sigma-Aldrich; Celltracker Green CMFDA and Red CM-Dil vital dyes were purchased from Life Technologies (Carlsbad, CA, USA).