Table 1.
Characteristic of primary and secondary antibodies and visualization systems used in IHC staining.
| Antibody (vendor) | Primary antibody dilution* | Secondary antibody (vendor) | Chromogen (vendor) | Positive control |
|---|---|---|---|---|
| Mouse anti-HIF-1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-53546) | 1:750** | EnVision+System HRP Labelled Polymer (Dako, Glostrup, Denmark, Cat# K4000) | Vector NovaRED HRP substrate (Vector Laboratories, Inc., Burlingame, CA, USA, Cat#SK-4800) | breast cancer |
| Rat anti-VDR (Abcam, Cambridge, MA, Cat#ab8756) |
1:75 | Vectastain ABC-AP Kit (Vector Laboratories Inc, Burlingame, CA, Cat#AK-5004) | Vectastain ABC-AP Reagent and alkaline phosphatase substrate (Vector Laboratories Inc, Burlingame, CA, USA, and SK5100). | normal skin/kidney |
| Rabbit anti-CYP27B1 (Santa Cruz Biotechnology, Santa Cruz, CA, Cat#sc-67261) | 1:75 | EnVision+System HRP Labelled Polymer (Dako, Glostrup, Denmark, Cat# K4002) | ImmPACT NovaRED (Vector Laboratories Inc., Burlingame, CA, USA, Cat#SK-4805) | kidney |
| Rabbit anti-CYP24A1 (Abcam, Cambridge, MA) |
1:40 | EnVision+System HRP Labelled Polymer (Dako, Glostrup, Denmark) | ImmPACT NovaRED (Vector Laboratories Inc., Burlingame, CA, USA) | kidney |
| Rabbit anti-RORγ (Dr Anton M. Jetten) | 1:50 | EnVision™ FLEX/HRP (Dako, Glostrup, Denmark, Cat# K4002) | Vector NovaRED HRP substrate (Vector Laboratories, Inc., Burlingame, CA, USA, Cat#SK-4800) | normal skin |
| Goat-antiROR α (Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat#sc-6062) | 1:25 | Vectastain Elite ABC Goat IgG (Vector Laboratories, Burlingame, CA, USA, Cat#PK6105) | ImmPACT™ NovaRED™ HRP substrate (Vector Laboratories, Cat#SK-4805) | normal skin |
| Mouse anti-FoxP3 (Abcam, Cambridge, UK, Cat#ab20034) | 1:100 | EnVision+System HRP Labelled Polymer (Dako, Glostrup, Denmark, Cat# K4000) | Vector NovaRED HRP substrate (Vector Laboratories, Inc., Burlingame, CA, USA, Cat#SK-4800) | lymph node |
ON – overnight;
*
primary antibodies were applied overnight at 4°C;
**
Tyramide Signal Amplification kit (Life Technologies, Carlsbad, CA, Cat#T20931) was used according the manufacturer’s protocol