Pt link device

Sagittal cross-sections were deparaffinized in xylene and rehydrated with alcohol gradients and demineralized water. The sections were stained for GFAP, IBA-1, CD68 and triggering receptor expressed on myeloid cells 2 (TREM2) as detailed in Table 1. Antigen retrieval was performed by heat in a water bath (96°C, 40 min) for GFAP and IBA-1 immunostaining or in citrate buffer (pH 6, 96°C, 20 min) using a Dako PT-link device (Dako, Glostrup, Denmark) for CD68 and TREM2 immunostaining.

Freshly cut TMA sections were all stained for CD8 in one run on the same day. The slides were deparaffinized, rehydrated, exposed to heat-induced antigen retrieval for 15 min at 98 °C in pH 9 DAKO target retrieval Solution (S2367) using a DAKO PT-LINK device, and then transferred to a DAKO Link 48 autostainer device. The autostainer protocol includes peroxidase blocking for 5 min (DAKO, Envision Flex-Kit 8002) and subsequent incubations with the primary antibody (Oncodianova, mouse monoclonal antibody, Clone TC8, dilution 1:200) for 20 min at room temperature, Flex HRP (DAKO Envision Flex-Kit 8002) for 20 min, DAB-Chromogen (DAKO Envision Flex-Kit 8002) for 10 min, and a final incubation with Hämatoxylin (DAKO K8008) for 5 min.

Splenic tissue was fixed in 10% neutral buffered formalin and then embedded in paraffin. To remove paraffin and rehydrate splenic sections a PT Link device (DAKO, Stockport UK) was used according to the manufacturer's instructions. Immunohistochemical staining was performed using a DAKO Auto-stainer automated slide processing system and the EnVision™ FLEX/HRP kit (DAKO, K8012). The primary antibodies used were PKCβII (C-18) and HA (C29F4). Hematoxylin and eosin (H&E) staining of splenic sections was carried out according to standard protocols.