Envision flex kit

Freshly cut tissue sections were immunostained in a DAKO Link 48 autostainer device. Slides were deparaffinized and exposed to heat-induced antigen retrieval for 10 min at 98 °C in pH 9 Tris-EDTA-Citrate buffer. Primary antibody specific for PD-L1 (E1L3N, CST, San Diego, CA, USA, 1:200) was applied at room temperature for 20 min. Bound antibody was then visualized using the EnVision Flex Kit (DAKO, Glostrup, Denmark) according to the manufacturer’s directions.
Complete absence of staining was regarded as “negative”. Staining of any intensity found in more than 1% of tumor cells was considered positive and the percentage of PD-L1⁺ tumor cells was assessed as described [51 (link)].

Prior to immunohistochemical staining of p16 EDTA, buffer pH 9 (Dako EnVision Flex Target Retrieval Solution high pH; K 8004; lot 20062462) was used for heat-induced epitope retrieval. Staining was performed by using an automated staining system (DAKO Autostainerplus) and Envision Flex Kit (Dako EnVision TM FLEX HRP/Dab; K 8010) according to manufacturers’ instructions. The following antibodies were used: p16 CINtec Histology Kit (Roche, Basel, Switzerland, #E6H4) for 30 min. An avidin-biotin-complex peroxidase technique, using aminoethylcarbazole for visualization and hematoxylin for counterstaining, was used.

Rats were anesthetized with sodium pentobarbital and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed and postfixed in the same fixative solution for 24 h at 4 °C. Paraffin-embedded sections (5-μm thick) from the rats or humans were cut and mounted on coated glass slides, processed with the Envision Flex+ Kit (Dako) to block endogenous peroxidase activity for 5 min, and incubated with an anti-Iba-1 (1:300; 30 min; Wako), anti-CD-68 (1:100; 60 min; Abcam), anti-GFAP (ready-to-use; 20 min; Dako), or anti-TNF-a (1:2000; 45 min; Abcam) antibody. The reaction was visualized by incubation with Envision Flex+ horseradish peroxidase for 20 min and then with diaminobenzidine for 10 min. The sections were counterstained with Mayer’s hematoxylin for 5 min.

TMA blocks were cut into 4-µm sections. IHC reactions were performed using the Dako Autostainer Link48 (Dako, Glostrup, Denmark). Deparaffinisation, rehydration, and epitope retrieval (97°C, 20 min) were performed using a low pH Target Retrieval Solution (Dako/Agilent Technologies, Santa Clara, CA, USA) in a PT- Link (Dako). Subsequently, the sections were washed in Tris-buffered saline and incubated with primary antibodies at room temperature for 20 min. The following specific primary antibodies were used: polyclonal rabbit anti-Periostin (dilution 1:200; code no.NBP1-82472; Novus Biologicals, Littleton, CO, USA), monoclonal mouse anti-Ki-67 antibody (ready-to-use, Clone MIB-1, code IS626; Dako), anti-TTF-1 (ready-to-use, Clone 8G7G3/1, code IR056; Dako), anti-p63 (ready-to-use, Clone DAK-p63, code IR662; Dako), anti-Podoplanin (ready-to-use, clone D2-40 (PDPN), code ISO072; Dako), anti-Vimentin (ready-to-use, clone V9, code GA630; Dako), and anti-αSMA (ready-to-use, clone IS611, code 1A4; Dako). The sections were then visualized using an EnVision FLEX kit (Dako). All slides were counterstained with haematoxylin (Dako). Negative control sections were generated in the absence of the primary antibody.

Immunohistochemical studies were performed using a rabbit polyclonal antibody against mPGES-1 (ref. HPA045064 prestige antibodies, diluted 1 : 50) from Sigma and a mouse monoclonal antibody anti-EP-4 (ref. 101775, diluted 1 : 100) from Cayman Chemical. Blanks were performed using the corresponding blocking peptides all from Cayman. Monoclonal antibodies (ref. M0616, diluted 1 : 35; ref. IR751 and ref. IR613, without further dilution) from Dako were used for von Willebrand Factor (vWF, endothelial cell marker), CD45 (pan-leukocyte marker), and CD68 immunostaining. Three-micrometer sections of paraffin-embedded tissue samples were stained in a Dako Autostainer Link 48 using the Dako EnVision Flex kit. Diaminobenzidine was used as chromogen. Immunostainings used for comparative purposes were processed simultaneously.

The TMA slides were dewaxed and washed. Prior to immunohistochemical staining, a citrate buffer with pH 6 (Dako EnVision Flex Target Retrieval Solution low pH; DM829; lot 20048823, Agilent Technologies, Santa Clara, CA, USA) was used for heat-induced epitope retrieval. Staining was performed by using an automated staining system (DAKO Autostainer^plus) and Envision Flex Kit (Dako EnVision TM FLEX HRP/Dab; K 8010), according to manufacturers’ instructions. The following antibodies were used: p4EBP1 (T37/46) (Cell Signaling Technology, Danvers, MA, USA, #2855), pp70S6K (T389) (Cell Signaling Technology, #9206), pS6 (S235/236) (Cell Signaling Technology, #4858), pPRAS (T246) (Cell Signaling Technology, #2997), pmTOR (Cell Signaling Technology, #2976), panAKT (Cell Signaling Technology, #4691), S6K1 (Abcam, Cambridge, UK, # ab32359); all incubated for 30 min and mTOR (Cell Signaling Technology, #2983), and pAKT (S473) (Dako, #S473); both were incubated for 60 min. An avidin-biotin-complex peroxidase technique, using aminoethylcarbazole for visualization and hematoxylin for counterstaining, was applied. Positive and negative controls were performed. pRaptor (Ser792) (Cell Signaling Technology, #2083) was very weakly expressed in the PeCa tissue and therefore was excluded from further analysis.