Diaminobenzidine substrate

Animals were transcardially perfused with 4% formalin (Chempur, Piekary Slaskie, Poland). The CNS and spleen were removed, post-fixed for 2 days in 4% formalin solution, and embedded in paraffin. Sections (10 µm thick) were placed onto glass slides (Menzel-Glaser, Braunschweig, Germany). Slides with paraffin-embedded sections were deparaffinized by immersion in xylene (Sigma, Poznan, Poland), followed by rehydration in ethanol (POCH, Gliwice, Poland). Antigen retrieval was done with 10 mM citrate buffer, pH 6.0 (Sigma, Poznan, Poland). Tissue sections were incubated overnight at 4°C, with primary rat anti-Foxp3 antibody (eBioscience Inc., San Diego, CA, USA) at 1:500 dilution. Secondary biotinylated rabbit anti-rat antibodies were purchased from DAKO North America Inc. (Carpinteria CA, USA) and ABC kit from Vector Laboratories (Peterborough, United Kingdom). Staining was performed using diaminobenzidine substrate (Sigma, Poznan, Poland) and counterstaining was done with hematoxylin-eosin (Sigma, Poznan, Poland).

The tissue cross-sections were labeled with mouse monoclonal anti-CHOP (1:300; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 2895), mouse monoclonal anti-GFP (1:300; Cell Signaling Technology; cat. no. 2955), and goat monoclonal anti-vaspin (1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-79815) at 4°C overnight, following microwave antigen retrieval in citrate buffer. After washing with PBS with Tween 20 (PBST), the tissue samples were incubated with rabbit anti-mouse (1:200; Gene Tech Biotechnology Co., Ltd., Shanghai, China; cat. no. GK500705) and donkey anti-goat secondary antibodies (1:200; Santa Cruz Biotechnology, Inc.; cat. no. sc-2042) at 37°C for 1 h, and then incubated with diaminobenzidine substrate (0.05%; Sigma-Aldrich) for 1–3 min. Specimens were counter-stained with hematoxylin and images were captured under a Leica DMI6000 microscope. Three sections from each animal were stained for lesion quantification, which was expressed as a percentage of total lesion area. Data are presented as the mean ± standard error of the mean (SEM).

All tissues were fixed in 10% formalin, embedded in paraffin, and cut into 5‐μm sections. The sections were deparaffinized in xylene and rehydrated in graded concentrations of ethyl alcohol. Antigen retrieval was performed by heating the sections in a microwave oven for 10 minutes with .01 mol/L citrate buffer (pH 6.0). Endogenous peroxidases and nonspecific reactions were blocked with 3% hydrogen peroxide and 5% BSA for 30 minutes, respectively. The sections were then incubated with an anti–REV7 antibody (Abcam, Cambridge, UK) at a dilution of 1:100 overnight. Labeling for REV7 was detected using biotinylated secondary antibodies, visualized with diaminobenzidine substrate (Sigma‐Aldrich, St Louis, MO, USA), and counterstained with hematoxylin (Sigma‐Aldrich). All steps were performed at room temperature. As a negative control, PBS was used in the absence of the primary antibody, confirming the specificity of this antibody. Immunostained slides were evaluated by 2 independent observers using light microscopy in a blinded manner. Immunohistochemistry (IHC) images were taken using an Olympus microscope. aScores were evaluated in a range of 0‐3 points at intervals of .2 points. Counts of IHC staining (Ki67 and CD31) were performed in images at a magnification of ×20, and cases were regarded as positive only if both observers independently defined them as such.