TABLE 2.
Immunohistochemical Protocols Used in the Analysis
| IHC | Primary antibody (Table 1) | Secondary antibody | Tertiary step | Quaternary step |
|---|---|---|---|---|
| CRABPII | CRABPII (1:300) | b-DαG1,2 | ABC3→DAB4 | |
| RALDH-1 | RALDH-1 (1:1000) | FITC-DαRb | ||
| RALDH-2 | RALDH-2 (1:1000) | FITC-DαRb | ||
| RALDH-2 + | RALDH-2 (1:1000) | FITC-DαRb | Rabbit αFITC | FITC-DαRb |
| CD31 | CD31 (1:50) | TxR-DαR | ||
| RALDH-2 + | RALDH-2 (1:10K) | b-DαRb | 5 TSA→6 FITC-SA | |
| CD54 | CD54 (1:20) | TxR-GαH | ||
| RALDH-2 + | RALDH-2 (1:500) | b-DαRb | TxR-SA | |
| F4/80 | F4/80 (1:100) | FITC-DαR | ||
| RALDH-2 + | RALDH-2 (1:1000) | b-DαRb | TxR-SA | FITC-DαM |
| OCAM | OCAM (1:50) | FITC-DαG | Mouse αFITC | |
| RALDH-2 + | RALDH-2 (1:10K) | b-DαRb | TSA→FITC-SA | TxR-DαRb |
| p75 | p75 (1:100) | TxR-DαRb | Rabbit αTxR | |
| RALDH-2 + | RALDH-2 (1:10K) | b-DαRb | TSA→TxR-SA | FITC-DαRb |
| PGP9.5 | PGP9.5 (1:1000) | FITC-DαRb | Rabbit αFITC | |
| RALDH-2 + | RALDH-2 (1:1000) | FITC-DαRb | Rabbit αFITC | FITC-DαRb |
| RALDH-3 | RALDH-3 (1:10K) | |||
| b-DαRb | TSA→TxR-SA | |||
| RALDH-2 + | RALDH-2 (1:1000) | b-DαRb | TSA→TxR-SA | |
| Vimentin | Vimentin (1:10) | FITC-DαG | ||
| RALDH-3 | RALDH-3 (1:1000) | FITC-DαRb | ||
| RALDH-3 + | RALDH-2 (1:1000) | FITC-DαRb | Rabbit αFITC | FITC-DαRb |
| CD31 | CD31 (1:50) | TxR-DαR | ||
| RALDH-3 + | RALDH-3 (1:10K) | b-DαRb | TSA→FITC-SA | |
| CD54 | CD54 (1:20) | TxR-GαH | ||
| RALDH-3 + | RALDH-3 (1:500) | b-DαRb | TxR-SA | |
| F4/80 | F4/80 (1:100) | FITC-DαR | ||
| RALDH-3 + | RALDH-3 (1:10K) | b-DαRb | TSA→TxR-SA | FITC-DαRb |
| GFP | GFP (1:5000) | FITC-DαRb | Rabbit αFITC | |
| RALDH-3 + | RALDH-3 (1:1000) | b-DαRb | TxR-SA | FITC-DαM |
| OCAM | OCAM (1:50) | FITC-DαG | Mouse αFITC | |
| RALDH-3 + | RALDH-3 (1:10K) | b-DαRb | TSA→TxR-SA | FITC-DαRb |
| PCNA | PCNA (1:1000) | FITC-DαRb | Rabbit αFITC | |
| RALDH-3 + | RALDH-3 (1:10K) | b-DαRb | TSA→FITC-SA | TxR-DαRb |
| p75 | p75 (1:100) | |||
| TxR-DαRb | Rabbit αTxR | |||
| RALDH-3 + | RALDH-3 (1:10K) | b-DαRb | TSA→TxR-SA | FITC-DαRb |
| PGP9.5 | PGP9.5 (1:1000) | FITC-DαRb | Rabbit αFITC | |
| RALDH-3 + | RALDH-3 (1:1000) | b-DαRb | TSA→TxR-SA | |
| Vimentin | Vimentin (1:10) | FITC-DαG |
b-DαG, biotinylated-donkey antigoat IgG; b-DαRb, biotinylated-donkey antirabbit IgG; FITC-DαG, FITC conjugated-donkey antigoat IgG; FITC-DαRb, FITC conjugated-donkey antirabbit IgG; FITC-DαR, FITC conjugated-donkey antirat IgG; TxR-DαG, Texas Red conjugated-donkey antigoat IgG; TxR-DαRb, Texas-Red conjugated-donkey antirabbit IgG; TxR-DαR, Texas-Red conjugated-donkey anti-rat IgG; TxR-GαH, Texas Red conjugated-goat anti-hamster IgG.
All secondary antibodies were used at a 1:50 dilution.
ABC, avidin (1:50) + biotinylated horseradish peroxidase (1:50).
DAB, 3,3′ diaminobenzedine.
TSA, Tyramide Signal Amplification Kit from Perkin-Elmer.
TxR-SA, Texas-Red conjugated-streptavidin; FITC-SA, FITC conjugated-streptavidin.