Novolink kit

Samples were fixed in 10% formalin for at least 24 h at room temperature (RT), processed and embedded in paraffin. Tissues were cut into 4 μm thick sections, dewaxed in xylene, and rehydrated with graded industrial denatured alcohol before staining as previously described [18 (link),21 (link)]. Histology was performed by staining the tissue sections with Harris’s Hematoxylin and Eosin (H&E) (Leica, Wetzlar, Germany), and Picro-Sirius Red (SR) solutions (Hopkin and Williams) (BDH Chemicals Ltd., Cellpath Ltd., Newtown, UK). When performing immunohistochemistry, antigen retrieval was achieved with sodium citrate buffer (pH 6.0) in a microwave (640 W) for 10 min. Nonspecific binding was blocked with normal horse serum (VECTASTAIN^®, Vector Laboratories, Peterborough, UK) for 5 min at RT. Sections were incubated for 1 h at RT with primary antibodies (see Supplementary Table S2). After washing, sections were incubated with pre-diluted biotinylated pan-specific universal secondary antibody (VECTASTAIN^®, Vector Laboratories, Peterborough, UK) for 30 min. Primary antibodies were detected with the Novolink™ Kit (Novocastra RE7280-K, Newcastle, UK). Slides were counterstained with hematoxylin, dehydrated, cleared, and mounted. Images were captured using the Axiocam IcC5.

Human colon adenocarcinoma tissue samples and adjacent normal tissue samples that had been fixed in formalin and embedded in paraffin previously were subjected to immunohistochemistry for PLIN2 (AP125; Research Diagnostics Inc., Flanders, NJ) and Ki-67 (MIB-1; Dako). Tissue-containing blocks were cut into 3-μm slices and were placed in slides treated with 4% (3-aminopropyl)triethoxysilane (APTS) (Thermo Scientific). Then the slides were incubated at 60°C for 16 h, followed by a deparaffinization protocol with five 3-min baths in absolute ethanol and one 10-min bath with water. Antigenic recovery occurred in a citrate solution (pH 6.0) at 98°C for 40 min for PLIN2 and in a pressure cooker with a citrate solution for 3 min for Ki-67. We used the Novolink kit (Novocastra) for signal amplification, following the manufacturer’s instructions. Primary antibodies were incubated for 12 h at 4°C, and after staining, the slices were counterstained with hematoxylin for 30 s, dehydrated in five baths with absolute ethanol, and embedded in five xylol baths. The coverslips were then mounted using Entellan mounting medium (Merck). Immunohistochemistry slides were evaluated independently by three different pathologists.

Laryngeal tumor biopsies from 58 patients with advanced laryngeal squamous cell carcinoma were used in this study. The patients participated in a phase III randomized trial where they received accelerated radiotherapy with or without carbogen gas and nicotinamide.⁶ (link) 79 patients participated in a multicenter translational side study and received PIMO intravenously two  hours before biopsy. In this present study, the tissue of 58 patients who were included in the RadboudUMC, Nijmegen, the Netherlands (single-center) was used.
Consecutive sections were cut from each tissue block and immunohistochemically stained for two hypoxic markers, HIF-1α, and PIMO. The staining procedure was done as previously described.³ (link) For PIMO, we used the primary antibody Mouse-antiPIMO (Lot# 9.7.11, HydroxyProbe, Massachusets, USA). For the HIF-1α IHC, the Novolink kit (Leica Biosystems, Rijswijk, the Netherlands) was used with the primary antibody Mouse-anti-HIF-1α (BD Biosciences, cat# 610959, lot 4 073 775).

Representative paraffin‐embedded tissue blocks of BC tissue were retrieved and processed using a protocol for the dual H&E and IHC staining; 4‐μm tissue sections were cut onto charged slides, and then placed on a 60°C hotplate for 20 min. After rehydration, slides were submerged in citrate buffer at pH 6.0. Water bath heat‐assisted retrieval for 30 min at 96°C was applied with citrate buffer.
Rabbit polyclonal anti PHH3 (Abcam, Cambridge, MA, USA; phospho S10 antibody, ab5176) was diluted at 1:100 in Leica antibody diluent (RE AR9352, Leica, Biosystems, Newcastle upon Tyne, UK) and incubated with the sections for 60 min at room temperature. The DAB (Novolink kit, Leica, Biosystems) working solution was applied. Haematoxylin nuclear stain was applied for a longer period (8 min), to remove nonspecific background staining and to improve contrast, weak acid alcohol was used, and then eosin counterstain was applied (2 min); Figure 1. Tonsil tissue was used as a positive control.
Stained slides were scanned at 40× magnification using a high‐throughput slide scanner (Pannoramic 250 Flash III; 3DHistech, Budapest, Hungary), and the slides were then viewed with case viewer software program (v. 2.2.0.85; 3D‐Histech).