Mouse anti human cd8 clone c8 144b

Microarray slides composed of formalin-fixed paraffin-embedded ovarian tumour cores were dewaxed and rehydrated prior to heat-induced epitope retrieval using a pressure cooker and a citrate-based antigen unmasking solution (Vector Laboratory). Detection of CD8⁺ T cells, CD45RO⁺ memory lymphocytes and CD68⁺ macrophages was performed using the mouse anti-human CD8 (clone C8/144B, Dako), mouse anti-human CD45RO (clone UCLH, Dako) and mouse anti-human CD68 (clone M0876, Dako) antibodies, using ultrasensitive Polymer-HRP IHC Detection system (Biogenex). Immunohistochemical protocols and slide hybridisations were carried out manually. Sections were counterstained with haematoxylin and mounted with DPX mounting medium (Sigma). Previously published PTEN immunostaining data was used where high PTEN expression was considered to be positive staining and low expression to be weak, heterogeneous or negative staining, respectively.^{21 (link)}

Tissue microarray blocks were sectioned and placed on SuperFrost Plus slides (Fisher Scientific, Waltham, MA, USA). After deparaffination and dehydration of sections, antigen retrieval was performed by boiling sections for 40 min in DAKO Target Retrieval Solution (TRS) pH = 6 using a water bath (Glostrup, Denmark). Next, sections were blocked for endogenous peroxidase with 0.3% H₂O₂ in PBS and additionally blocked using 5% BSA in PBS. The primary antibody against FTH1 (rabbit anti-human FTH1, clone EPR3005Y; Genetex, Irvine, CA, USA) was diluted 1:100 in DAKO Antibody Diluent and incubated for 1 h at room temperature. FTH1 expression was visualized using the rabbit Envision+ system (DAKO), and nuclei were counterstained with hematoxylin.
FFPE blocks from 51 of the 241 BRCA1/2 (42 BRCA1 and 9 BRCA2) mutation carriers that were retrieved from the Erasmus University Medical Center were additionally stained for leukocyte marker CD45, cytotoxic T-cell marker CD8, helper T-cell marker CD4, and regulatory T-cell marker FOXP3 on whole sections, as described above. Antibodies were mouse anti-human CD45 clone 2B11 and PD7/26 (Cell Marque (Rocklin, CA, USA); TRS pH = 8, ready-to-use), mouse anti-human CD8 clone C8/144B (DAKO; TRS pH = 9, 1:100), mouse anti-human CD4 clone 4B12 (DAKO; TRS pH = 9, 1:80), and mouse anti-human FOXP3 clone 236A/E7 (Abcam (Cambridge, UK); TRS pH = 6, 1:50).