Table 1.
Reagents for immunostains on mouse tissue
| Immunostain | Antigen retrieval | Block | Primary antibody | Secondary antibody | Tertiary reagent |
|---|---|---|---|---|---|
| Thrombomodulin (CD141) | Citrate buffer 30 min in steamer (Vector H-3300); rinses with TBST | 10% horse serum in 1∶1 5% BSA∶Superblock (ScyTek AAA5000) 1 hr at RT | 1∶1,000 1 hr at RT (R&D Systems AF3894) | 1∶200 AF594 donkey anti-goat (Invitrogen A11058) 1 hr at RT | Vectashield with DAPI (Vector H-1200) |
| α–smooth muscle actin | Citrate buffer 30 min in steamer (Vector H-3300); rinses with TBST | Avidin 10 min, Biotin 10 min, mouse-on-mouse (MOM) kit blocking solution (Vector BMK-2202) 1 hr at RT | 1∶100 30 min at RT (Dako M0851) | MOM biotinylated anti-mouse reagent (Vector BMK-2202) 10 min at RT | Texas Red-streptavidin 1∶2,000 (Invitrogen S872), Vectashield with DAPI (Vector H-1200) |
| PCNA | Citrate buffer 30 min in steamer (Vector H-3300); rinses with TBST | 10% horse serum in 1∶1 5% BSA/Superblock | 1∶50 1 hr at RT (Santa Cruz scbt 7907) | 1∶200 1 hr at RT AF488 donkey anti-rabbit (Invitrogen A21206) | Vectashield with DAPI (Vector H-1200) |
| IL-4 | Citrate buffer 30 min in steamer (Vector H-3300); rinses with TBST | Peroxide block (Dako S2003) 10 min, 3% H2O2 in PBS 10 min, avidin 10 min, biotin 10 min, 10% goat serum in TBS 1 hr at RT | 1∶100 1 hr at RT (Abcam ab9622) in TBS | 1∶200 1 hr at RT biotinylated goat anti-rabbit (Vector BA5000) in TBS | SA-HRP 30 min at RT (Vector SA-5704), DAB (Vector SK-4100) 5 min at RT, hematoxylin 2 min at RT |
| IL-13 | Citrate buffer 30 min in steamer (Vector H-3300); rinses with TBST | Peroxide block (Dako S2003) 10 min, 3% H2O2 in PBS 10 min, avidin 10 min, biotin 10 min, 10% rabbit serum in TBS | 1∶50 1 hr at RT (R&D Systems AF-213-NA) in TBS | 1∶200 1 hr at RT biotinylated rabbit anti-goat (Vector BA1000) in TBS | SA-HRP 30 min at RT (Vector SA-5704), DAB (Vector SK-4100) 5 min at RT, hematoxylin 2 min at RT |
| TUNEL | Manufacturer’s instructionsa | Manufacturer’s instructionsa | Manufacturer’s instructionsa | Manufacturer’s instructionsa | Manufacturer’s instructionsa |
Note
TBST: Tris-buffered saline plus Tween; BSA: bovine serum albumin; RT: room temperature; DAPI: 4′,6-diamidino-2-phenylindole; PCNA: proliferating cell nuclear antigen; PBS: phosphate-buffered saline; TBS: Tris-buffered saline; SA-HRP: horseradish peroxidase streptavidin; DAB: diaminobenzadine; IL: interleukin; TUNEL: terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling;
a
Promega Dead End TUNEL Kit (catalog no. G3250).