Dako autostainer

Human coronary arteries were fixed and paraffin embedded. De-paraffinized sections were treated with target retrieval solution DAKO citrate pH 6.1 (Agilent, Santa Clara, CA). Slides were labeled with anti-ADAMTSL4 (Sigma, St. Louis, MO) on the DAKO Autostainer (Agilent, Carpinteria, CA) using ImmPRESS Rb (Vector Labs, Burlingame, CA) and diaminobenzadine as the chromogen. Slides were imaged on EVOS microscope (ThermoFisher, Waltham, MA).
In situ hybridization for ADAMTSL4 mRNA was performed on a normal human coronary artery sample. Five-micron sections were obtained from the fixed and paraffin-embedded human coronary artery, and slides were processed according to the RNAscope Multiplex Fluorescent V2 assay manufacturer’s instructions (ACDbio, Newark, CA). The probes used were as follows: C1- Hu TAGLN, C2-Hu ADAMTSL4, C3-HU ACTA2. Opal fluorophores (520, 570, 690) were used at 1:1000 dilution (Akoya Biosciences, Marlborough, MA). Slides were imaged on Nikon Ti microscope using the Element Software (Nikon, Melville, NY).

Formalin-fixed paraffin-embedded brain sections (4 μm thick) were incubated with primary antibodies after heat mediated antigen retrieval. All immunohistochemical procedures were performed following preprogramed standard protocol on an automatized histostainer (Dako Autostainer, Agilent) with positive and negative controls in each operation. Immunoreactivity was visualization with diaminobenzidine and magenta chromogens (Dako EnVision). A staining index was calculated as the product of intensity and fraction of positive tumor cells using Aperio ImageScope (Leica)⁵¹ . The immunoreactivity of human brain tissues were scored by two board-certified pathologists as previously described^{52 (link)}. Primary antibodies were: EGFR (prediluted, Thermo Scientific, RM-2111-RQ), Ki-67 (prediluted, Dako, GA62661-2), Claudin-5 (1:500, Thermo Scientific, 34–1600) and ERG (1:1000, Abcam, EPR3864). Secondary antibody: EnVision FLEX + rabbit (linker) (prediluted, Dako, SM805) for EGFR; no secondary antibody was used for all other antibodies.

Immunohistochemistry for cytoplasmic CD3 and nuclear PAX5 antigens were performed at the Animal Health Laboratory, University of Guelph (an American Association of Veterinary Laboratory Diagnosticians accredited laboratory), using an automated staining instrument (Dako Autostainer, Dako/Agilent, Ontario, Canada). The immunohistochemical reaction for CD3 followed the protocol detailed by Hall et al. (12 (link)). For PAX5, after manual deparaffinization and rehydration, the sections were treated with 3% hydrogen peroxide to quench endogenous peroxidase activity. Heat-induced epitope retrieval was carried out using an EDTA buffer (pH 9) and a pressure cooker device (PT Link, Dako). Primary antibodies against PAX5 (mouse monoclonal, clone 24, Biocare Medical) were diluted 1:50, and incubated at RT for 30 mins. Slides were then incubated with a dual anti-mouse / anti-rabbit anti-IgG horseradish peroxidase–linked polymer (EnVision FLEX, Dako) for 30 mins at RT, and reactions were visualized using Nova Red chromogen (Vector Laboratories, Burlington, ON, Canada). Slides were counterstained with hematoxylin. For CD3 and PAX5 immunohistochemical analysis, thymus, and bursa of Fabricius were used as positive controls, respectively. Negative controls were incubated in the absence of primary antibody. Sections were imaged using the same microscope and camera as described previously.

All dissected LNs were fixed in formalin, embedded in paraffin, and cut into 3-mm transverse sections. After deparaffinization and rehydration, 4- to 5-µm sections were stained with hematoxylin-eosin and examined by one of three pathologists with high experience in urological malignancies. If conventional histology was inconclusive, immunohistochemistry with a pancytokeratin antibody (AE1/AE3) was carried out using a DAKO Autostainer (Agilent Technologies, Santa Clara, CA, USA). In cases of false-negative DSNB results, SLNs were histopathologically reexamined by one pathologist.