Anti cd45r

Immunohistochemistry (IHC) was performed as described previously (22 (link)) applying the avidin-biotin-peroxidase (ABC) complex method and using antibodies for the detection of TBEV (anti-TBEV E protein clone 1493, Matthias Niedrig, mouse monoclonal), T cells (anti-CD3, Agilent Dako, Cat.No. A0452, rabbit polyclonal), B-lymphocytes (anti-CD45R, BD Bioscience, Cat.No. 553085, rat monoclonal), microglia/macrophages (anti-ionized calcium-binding adapter molecule 1 (Iba1), Wako Chemicals, 019-19741, polyclonal rabbit) and astrocytes (anti-glial fibrillary acidic protein (GFAP), Dako Cytomation, Cat.No. Z0334, rabbit polyclonal). Briefly, sections of brain and intestine were dewaxed and rehydrated in a graded series of alcohol. For anti-CD3, anti-CD45R and anti-Iba1 antibodies, antigen retrieval was achieved by boiling sections in citrate buffer (pH = 6) in a microwave (800W) prior to blocking of unspecific binding sites. For anti-TBEV and anti-GFAP antibodies, no pretreatment was necessary. After overnight incubation of primary antibodies, sections were incubated with the respective biotinylated secondary antibodies for 45 min. The staining was visualized using chromogen 3,3’-diaminobenzidine tetrahydrochloride (DAB) and counterstaining of nuclei with Mayer’s hematoxylin (Roth C.GmbH & Co KG).

For staining of Lin⁻/Sca-1⁺/c-Kit⁺ (SKL cells), Lin⁻/Sca-1⁺/CD45⁺ (HSCs), Sca-1⁺/Lin⁻/CD45⁻ (VSELs), Lin⁻/CD45⁻/CD31⁺ (EPCs), and Lin⁻/CD45⁻/CD31⁻/CD90⁺ (MSCs), the following monoclonal antibodies were used: FITC–anti-CD117 (also known as c-Kit, clone 2B8; BioLegend, San Diego, CA, USA) and PE–Cy5–anti-mouse Ly-6 A/E (also known as Sca-1, clone D7; eBioscience, San Diego, CA, USA). All anti-mouse lineage marker antibodies, including anti-CD45R (also known as B220, clone RA3-6B2), anti-Ter-119 (clone TER-119), anti-CD11b (clone M1/70), anti-T cell receptor β (clone H57–597), anti-Gr-1 (clone RB6-8C5), anti-TCRγδ (clone GL3), and anti-CD45 (clone 30-F11), conjugated with PE; anti-CD31 (clone MEC 13.3), conjugated with APC; and anti-CD90.2 (clone 30-H12), conjugated with BV510, were purchased from BD Biosciences. Staining was performed in RPMI-1640 medium containing 2% FBS. All monoclonal antibodies were added at saturating concentrations, and the cells were incubated for 30 min on ice, washed twice, and analyzed with a FACSVerse cytometer (BD Biosciences) [26 (link)].

HE staining and immunohistochemical staining was performed as previously described [26 (link)]. Briefly, for immunohistochemical staining, paraffin-embedded tissues, sectioned at 4 µm thickness, were dewaxed and boiled in Tris-EDTA buffer (10 mM Tris Base, 1mM EDTA-2Na, 0.05% Tween 20, pH 9.0) for 20 minutes. After blocking, anti-MRP8 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-MRP14 (Santa Cruz Biotechnology), anti-CD3 antibody (Santa Cruz Biotechnology) or anti-CD45R (BD Biosciences, San Jose, CA) was applied to the serial sections of tissues. After washing with PBS, sections were incubated with biotinylated anti-goat IgG (Nichirei Bioscience, Tokyo, Japan) or biotinylated anti-rat IgG (Nichirei Bioscience), followed by incubation with alkaline phosphatase-conjugated streptavidin (Nichirei Bioscience). Finally, enzymatic color development was performed by using 4-[(4-amino-m-tolyl) (4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-o-toluidine monohydrochloride (new fuchsine, Nichirei Bioscience). For quantitative analyses of infiltrating MRP14⁺ and MRP8⁺ cells in the tissues, the number of MRP14⁺ or MRP8⁺ cells in the immunohistochemically stained tissues was counted in 5 random microscopic fields at 400x magnification.

Antibodies included anti‐BrdU, ab1893 (Abcam, Cambridge, MA), heat‐induced epitope retrieval (HIER; pH 6.0), used at 1:500; anti‐Ki67, ab16667 (Abcam), HIER, 1:100; anti‐Mac‐3, #55322 (BD Pharmingen, San Diego, CA), HIER, 1:1000; anti‐CD3, A 0452 (DakoCytomation, Carpinteria, CA), HIER, 1:100; anti‐CD45R, #550286 (Pharmingen), HIER, 1:50; anti‐mannose receptor (MR), ab64693 (Abcam), HIER, 1:1000; anti‐YM1 (chitinase 3‐like 3), R&D Systems (Minneapolis, MN), HIER, 1:1000; anti‐iNOS (inducible nitric oxide synthase), ab15323, (Abcam), HIER, 1:100; anti‐arginase I (Arg I), BD Transduction, 1:100; anti‐FOXP3, FJK‐16 (eBioscience, San Diego, CA), HIER, 1:10; anti‐MPO (myeloperoxidase), 120‐15484 (Novus, Saint Charles, MO), HIER, 1:10; anti‐SMA (smooth muscle actin), A2547 (Sigma‐Aldrich), 1:200; anti‐CD68, clone KP1 (DAKO), HIER, 1:100; anti‐CD4, 14‐9766 clone 4SM95 (Affymetrix, Santa Clara, CA), HIER, 1:50; and anti‐CD8, 14‐0808 Clone 4SM15 (Affymetrix), HIER, 1:100. Isotype and preimmune negative controls were used during staining optimization and lacked background staining.

For the staining of Lin^–/Sca-1⁺/c-Kit⁺ (SKL) cells, the following monoclonal antibodies were used: FITC–anti-CD117 (also known as c-Kit, clone 2B8; BioLegend, San Diego, CA, USA), PE–Cy5–anti-mouse Ly-6 A/E (also known as Sca-1, clone D7; eBioscience, San Diego, CA, USA), and anti-mouse lineage-marker antibodies, including anti-CD45R (also known as B220, clone RA3-6B2), anti-Ter-119 (clone TER-119), anti-CD11b (clone M1/70), anti-T cell receptor β (clone H57-597), anti-Gr-1 (clone RB6-8C5), and anti-TCRγδ (clone GL3) conjugated with PE (BD Biosciences, San Jose, CA, USA). Staining was performed in RPMI-1640 medium containing 2% FBS. All monoclonal antibodies were added at saturating concentrations, and the cells were incubated for 30 min on ice, washed twice, and analyzed using an LSRII flow cytometer (BD Biosciences, San Jose, CA, USA) [4 (link), 7 (link), 26 (link)].