Cd68 clone ed1

Formalin-fixed paraffin-embedded pancreas tissues were serially sectioned. Consecutive sections were stained for H-E, insulin and glucagon, HA, CD68, CD3, and CD8 in different combinations. Four to six sets of serial sections were prepared per pancreas. All islets present in a stained section, on average 71 islets (range 52–99 islets), were counted per rat. The following primary antibodies were used: insulin (Dako, Glostrup, Denmark), glucagon (Sigma-Aldrich, St Louis, MI, USA), and CD68 (clone ED1, Bio-Rad, Hercules, CA, USA), at dilutions 1:500, 1:2,000, and 1:100, respectively. Antibodies to CD3 (clone SP7, Abcam, Cambridge, UK) and CD8 (Abcam) were used at dilution 1:150. Staining for HA was performed as described (16 (link), 18 (link)). Sections were incubated overnight with the primary antibodies and then for 1 h with the following secondary antibodies at dilution 1:400: Alexa Fluor^® 488 conjugated goat anti-rabbit IgG (Invitrogen), Alexa Fluor^® 568 conjugated goat anti-mouse IgG (Invitrogen), and Cy™2 donkey anti-guinea pig IgG (Jackson Immunoresearch). The stained sections were mounted with VECTASHIELD Vibrance with DAPI (a nuclear counterstain) Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA).

Immunohistology was performed on 3 μm thin, formalin-fixed, paraffin-embedded (FFPE) coronary sections using antibodies for macrophages/activated microglia (CD68, clone ED1, Bio-Rad AbD Serotec, Oxford, UK), CD3+ T-cells (clone CD3-12Bio-Rad AbD Serotec, Oxford, UK), CD45R+ B-cells (clone HIS24, BD Biosciences, Franklin Lakes, NJ, USA), myelin basic protein (MBP, Abcam, Cambridge, UK), anti-neuronal nuclei (NeuN, Abcam, Cambridge, UK), oligodendrocyte lineage factor 2 (OLIG2, clone 211F1.1, Merck Millipore, Darmstadt, Germany), mature myelin-maintaining oligodendrocytes (P25/TPPP, clone EPR3316, Abcam, Cambridge, UK), pre-resp. actively myelinating oligodendrocytes (BCAS1, Santa Cruz, Heidelberg, Germany), FITC (HRP conjugated, Dako Deutschland GmbH, Hamburg, Germany), and green fluorescent protein (GFP, clone 6AT316, Abcam, Cambridge, UK).

Antibodies against CD3 (clone F7.2.38; DAKO, Glostrup, Denmark) for T cells and CD68 (clone ED1; Bio-Rad, Hercules, CA, USA) for macrophages were used for immunohistochemistry. Deparaffinized sections were pretreated with microwave in Tris-EDTA buffer (pH 9.0) and with proteinase K (DAKO) (100 μg/mL, 37 °C, 10 min) for CD3 and CD68, respectively. Sections were then incubated with 5% skim milk in phosphate buffered saline (PBS) for 15 min and with primary antibody for 1 h, followed by 1 h incubation with peroxidase-conjugated secondary antibody (Histofine simple stain MAX PO; Nichirei Biosciences, Tokyo, Japan). Positive reactions were visualized with 3,3′-diaminobenzidine (DAB; Nichirei Biosciences; Tokyo, Japan). Sections were lightly counterstained with hematoxylin. In order to evaluate hepatocellular apoptosis, liver sections were subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method as previously described [19 (link),20 (link)]. The number of TUNEL-positive apoptotic hepatocytes was counted; more than 500 hepatocytes were analyzed in each animal to obtain reliably quantitative data.