Table 1.
Antibodies and Counterstains Used in This Study
| Cells | Epitope | 1°Antibody, Clone, Source, Dilution | 2°Antibody, Source, Dilution |
|---|---|---|---|
| Rat LSECs | Sinusoidal endothelial cell surface | IF and FACS: mouse anti-rat SE-1 (Dr. Katsuhiko Enomoto, Akita University, Akita, Japan; references 24, 25) 1:100 | IF: goat anti-mouse Cy3 (Jackson ImmunoLabs) 1:1000 or goat anti-mouse Alexa 488 (Invitrogen) 1:500 FACS: goat anti-mouse immunoglobulin G phycoerythrin (Invitrogen) 1:100 |
| Large vessel endothelial cells and LSECs, surface | Platelet-endothelial cell adhesion molecule | IF: Mouse anti-rat CD31 (Serotec) 1:100
FACS: Mouse anti-rat CD31 (Chemicon) 1:100 |
IF: goat anti-mouse Cy3 (Jackson ImmunoLabs) 1:1000 FACS: goat anti-mouse immunoglobulin G phycoerythrin (Invitrogen) 1:100 |
| All leukocytes | Common leukocyte antigen | IF: mouse anti-rat CD45, OX1 (Serotec) 1:100
FACS: mouse anti-rat CD45 conjugated to phycoerythrin, (BD PharMingen) 1:100 |
IF: goat anti-mouse Cy3 (Jackson ImmunoLabs) 1:1000 |
| Macrophages | Macrophage surface antigen | IF: mouse anti-rat ED2 (CD163) (Serotec) 1:100
FACS: ED2 (CD163)-phycoerythrin (Serotec) 1:10 |
IF: goat anti-mouse Cy3 (Jackson ImmunoLabs) 1:1000 |
| Macrophages/monocytes | Macrophage marker | IF: mouse anti-rat ED1 (CD68) (Serotec) 1:100 | IF: goat anti-mouse Cy3 (Jackson ImmunoLabs, West Grove, PA) 1:1000 |
| LEW cells | Lewis MHC class II | IF and ImmunoGold: mouse anti-Lewis rat MHC class II (L21-6) (Dr. Yuichi Iwaki, University of Pittsburgh, Pittsburgh, PA; reference 27) | IF: goat anti-mouse Cy3 (Jackson ImmunoLabs) 1:1000 ImmunoGold: goat anti-mouse15-nm gold conjugate (Amersham) 1:25 |
| BN cells | MHC class I | IF: mouse anti-BN MHC class I (OX27) (Serotec) 1:400 | IF: goat anti-mouse Alexa 488 (Invitrogen) 1:500 |
| VEGF | VEGF | IF: mouse anti-VEGF (Santa Cruz Biotechnology) 1:100 | IF: goat anti-mouse Alexa 488 (Invitrogen) 1:500 |
| Endothelial cells | VEGF-R2 | IF: rabbit anti-FLK-1 (Santa Cruz Biotechnology) 1:100 | IF: goat anti-rabbit Alexa 488 (Invitrogen) 1:500 |
| Endothelial cells | Endothelial cell scavenger receptor | IF: Di-I-acetylated LDL (BTI) 1:250 in culture medium for 4 hours before fixation | This is a vital dye that is endocytosed via the scavenger receptor and therefore is taken up only by live endothelial and macrophages in vitro |
| Apoptosis marker Counterstain | Annexin V Nucleus | FACS: annexin V-FITC (BD PharMingen)
Hoechst 33258: bis-benzimidizole (Sigma) 10 mg/mL PI (Sigma) 0.19 mg/mL |
Manufacturer locations are as follows: Amersham, Piscataway, NJ; BD PharMingen, San Diego, CA; BTI, Stoughton, MA; Chemicon, Temecula, CA; Invitrogen, Carlsbad, CA; Jackson ImmunoLabs, West Grove, PA; Santa Cruz Biotechnology, Santa Cruz, CA; Serotec, Raleigh, NC; Sigma, St. Louis, MO.
Abbreviation: IF, immunofluorescence; FACS, fluorescence-activated cell sorting.