Table 3.
Primary antibodies used for different immunohistochemical techniques and the retrieval methods applied for each of them.
| Antibody | Antigen | Type | Dilution | Retrieval Method | Source |
|---|---|---|---|---|---|
| L42 | Prion protein | Monoclonal | 1:500 | Formic acid 15 min Proteinase K 15 min Heat treatment 20 min Peroxidase blocking |
DAKO |
| Anti- GFAP | GFAP | Polyclonal | 1:500 | Peroxidase blocking | DAKO |
| Anti-IBA-1 | IBA-1 | Polyclonal | 1:1.000 | Heat treatment 20 min Peroxidase blocking |
WAKO |
| IL-1 alpha | IL-1 | Polyclonal | 1:100 | Autoclave 121 °C (citrate buffer 10%) | ThermoFisher |
| Anti-IL-1RN | IL-1R | Polyclonal | 1:100 | Autoclave 121 °C (citrate buffer 10%) | Sigma |
| IL-2R.1 | IL-2R | Monoclonal | 1:1.000 | PTLink 96 °C | ThermoFisher |
| 8H12 | IL-6 | Monoclonal | 1:20 | Autoclave 121 °C (citrate buffer 10%) | ThermoFisher |
| OTI1D10 | IL-10R | Monoclonal | 1:250 | PTLink 96 °C | ThermoFisher |
| Ber-H2 | TNFαR | Monoclonal | Ready to use | PTLink 96 °C | DAKO |
| IFNGR1 | IFNγR | Polyclonal | 1:200 | Autoclave 121 °C (citrate buffer 10%) | ThermoFisher |
Immunostained sections were scored by two independent operators on a scale ranging from 0 (minimum) to 4 (maximum), as previously described [14,19,20], regarding the intensity of pathological prion protein accumulation, and astroglial and microglial activation. Morphological glial alterations were also evaluated in 10 microscopic fields in each brain region. For neuroinflammatory markers, observers scored the intensity of immunostaining by counting positive cells [64] in 5 microscopic fields in each brain region examined.