Table 1.
Antibodies used for immunohistochemical analysis of ferroptosis-related markers and AD pathology in alphabetical order.
| Primary antibody | Secondary antibody | Tertiary antibody | ||||
|---|---|---|---|---|---|---|
| Target | Dilution | Manufacturer | Origin | Incubation | (Targtet, dilution (manufacturer)) | |
| Aβ (6F/3D) | 1:400 | DAKO, M087 | Mouse | 60 min, RT | I-view + Ampl | – |
| Cytochrome C | 1:500 | Abcam, ab90529 | Rabbit | 60 min, RT | GARPO, 1:100 (DAKO, P0448) | RAGPO, 1:100 (DAKO, P0449) |
| Ferritin | 1:50 | LSBio, B14992 | Rabbit | 60 min, RT | GARPO, 1:100 (DAKO, P0448) | RAGPO, 1:100 (DAKO, P0449) |
| Ferroportin | 1:50 | LSBio, B1836 | Rabbit | ON 4 °C | GARbio, 1:300 (DAKO, E0432) | StrepPO, 1:300 (DAKO, P0397) |
| GPX4 | 1:100 | Abcam, ab116703 | Goat | ON 4 °C | RAGbio, 1:300 (South. Biotech. Ass, 6165-08) | StrepPO, 1:300 (DAKO, P0397) |
| NCOA4 | 1:50 | Invitrigen, PA5-96398 | Rabbit | 60 min, RT | GARPO, 1:100 (DAKO, P0448) | RAGPO, 1:100 (DAKO, P0449) |
| AT-8/Tau | 1:20 | Invitrogen MN1020 | Mouse | 60 min, RT | Ultra IVIEW | – |
| 4HNE | 1:200 | Bioss, bs-6313R | Rabbit | 60 min, RT | GARPO, 1:100 (DAKO, P0448) | RAGPO, 1:100 (DAKO, P0449) |
AB antibody, Aβ amyloid beta, Ampl amplification, GARbio goat-anti-rabbit biotin, GARPO goat-anti-rabbit peroxidase, GPX4 glutathione peroxidase 4, min minute, NCOA4 nuclear receptor co-activator 4, ON overnight, RAGbio rabbit-anti-goat biotin, RAGPO rabbit-anti-goat peroxidase, RT room temperature of ~21 °C, StrepPO streptavidin peroxidase, 4HNE 4-hydroxy-2-nonenal.
Microwave treatment of three times 10 min in Tris/HCl buffer (pH 9) was used as the antigen retrieval method for all antibodies against ferroptosis-related markers. Three-minute treatment with 98% formic acid at RT was used as antigen retrieval method for Aβ antibody and no antigen retrieval method was used for tau. The diagnostic stainings, AT-8/Tau and Aβ (6F/3D), were stained on the Benchmark (Vetana) which did not require a tertiary antibody step.