Chromomap dab

For immunohistochemistry, 5 μm fresh-frozen tissue sections were fixed and stained with primary antibodies (CD68: 1:100; HLA-DR: 1:40; AIF1/IBA1: 1:500), secondary antibodies (Ultramap anti-mouse HRP multimer) and detection reagent (Ventana Chromomap DAB). Slides were processed on the Discovery Ultra platform (Ventana) and imaged using the ScanScope XT digital pathology slide scanner (Aperio).

Immunohistochemistry on lung adenocarcinoma tissue microarray was performed using PPAT mouse monoclonal antibody, PAICS mouse monoclonal antibody and PKM2 specific rabbit monoclonal antibody. Immunohistochemistry was performed using an automated protocol developed for the DISCOVERY XT automated slide staining system (Ventana Medical Systems, Inc.,) using Ultramap anti-mouse HRP or Ultramap anti-rabbit HRP as secondary antibody and was detected using ChromoMap DAB (Cat#760-159, Ventana Medical Systems Inc.,) Hematoxylin II (Cat#790-2208 Ventana-Roche, Tucson, AZ, USA) was used as the counterstain. Immunohistochemistry staining was evaluated by D.T. TMA sections were scored for the proportion of tumor cells staining for the marker and intensity using the methodology described by Harvey et al [70 (link)].

TMA paraffin block sections slides were dried overnight at 37°C. PRL-3 immunohistochemical staining of the cut sections was performed using the Ventana Discovery ULTRA autostainer (Tucson, Arizona, USA). After paraffin embedded tissues were dewaxed, antigen retrieval was applied using Ventana ULTRA Cell Conditioning-1 (CC1) for 32 minutes at 95°C. Endogenous peroxidase was blocked by discovery inhibitor CM (#760–4306, Ventana) for 12 minutes. Sections were incubated with non-commercial mouse monoclonal antibody[24 (link), 25 (link)] (kind gift from professor Qi Zeng, Agency for Science, Technology and Research (ASTAR), Singapore) with 1/50 dilution for 32 minutes at 36°C. As secondary antibody, OmniMap anti-mouse HRP (#760–4310, Ventana) was loaded for 20 minutes, followed by 8 minutes of HRP amplification. The detection chromogen was ChromoMap DAB (#760–159; Ventana). Counterstaining was performed using the hematoxylin II (#790–2208, Ventana) counterstain for 32 minutes and then with a bluing reagent for 8 minutes. Staining was performed in one single experiment and a human multiple organ (normal and malignant) tissue array was included for specificity control of antibody. Normal tonsil and liver adenocarcinoma were used as negative and positive tissue controls, respectively.

Immunohistochemistry (IHC) was performed on 4 µm thick FFPE tissue sections mounted on glass slides. All IHC steps were carried out on the Ventana Discovery XT automated platform (Ventana Medical Systems, Tucson, AZ). Sections were treated with Cell Conditioner 1, standard time, and then incubated in primary antibody against CD8 clone SP16 (Abcam, cat # ab101500) at 1:100 for 1 h at ambient temperature. Specifically bound primary antibody was detected by the OmniMap anti-rabbit HRP detection kit, followed by ChromoMap DAB (Ventana Medical Systems, Tucson, AZ). The sections were counterstained with Hematoxylin II (Ventana Medical Systems, Tucson, AZ), dehydrated, and coverslipped.