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Automated immunostainer

Manufactured by Agilent Technologies
Sourced in Denmark, United States

The Automated Immunostainer is a laboratory instrument designed for the automated processing of immunohistochemistry (IHC) and in situ hybridization (ISH) assays. The core function of this equipment is to automate the staining process, including antigen retrieval, primary antibody incubation, and chromogen detection, in a standardized and reproducible manner.

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24 protocols using automated immunostainer

1

Immunostaining of BM biopsies for NK cells

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Immunostaining of acetic acid-zinc-formalin fixed and paraffin embedded BM biopsy specimens was performed with anti-CD57 antibody on 4μm thick sections to identify NK cells. All reactions were performed using an automated immunostainer (DakoCytomation, Carpenteria, CA) in conjunction with the Envision-HRP detection system (Dako Cytomation) using diaminobenzidine as the chromogen. Both positive (normal tonsil) and negative (isotype-matched) controls were included. Nuclei were counterstained with hematoxylin. Sections were scanned using an Aperio ScanScope CS Digital image system and the images were processed by using Spectrum (Aperio Technologies, Vista, CA, USA).
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2

Immunohistochemical Profiling of Epigenetic Markers in FFPE Tissue Microarrays

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Sections from FFPE tissue samples were used to build TMAs. During pathological review of each section, areas containing malignant follicles, representative of the entire biopsy sample and avoiding fibrotic portions were marked on the paraffin blocks. Cylinders that were 1 mm in diameter from three different areas were then collected and included in the TMA blocks. After dewaxing and pressure-cooker antigen retrieval, immunostaining was conducted in an automated immunostainer (Dako, Glostrup, Denmark) using a standard avidin–biotin–peroxidase technique. The primary antibodies (EZH2, H3K27me2 and H3K27me3) and staining conditions were used as previously described.19 (link) Deparaffinization, rehydration, epitope retrieval and staining were performed as described by Dubois et al.19 (link) Slides were scored in a blinded fashion by three experienced anatomopathologists (LX, ST, LM). Tumors were scored according to the proportion of tumor cells stained (0–10, with 0 representing negative staining, 1 representing 1–10% positive tumor cells and 10 representing 91–100% positive tumor cells). For each patient, the methylation score was adapted from Dubois et al. as follows:

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3

Immunohistochemical Profiling of Vascular Pathology

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The immunohistochemical study was performed using an automated immunostainer (Dako A/S, Glostrup, Denmark), after heat-induced retrieval in citrate buffer with antibodies against α-smooth muscle actin (α-SMA, clone 1A4, 1/100), h-caldesmon (clone h-Cd, 1/100), CD45 (clone 2B11, 1/100), CD3 (polyclonal, 1/200), and CD68 (clone PGM1, 1/100) (Dako Omnis). The epitopes were detected using the Envision+ system horseradish peroxidase detection kit and revealed with liquid diaminobenzidine (Dako Omnis).
α-SMA and h-caldesmon stains were used to quantify the percentage of the fibrous rupture of the tunica media of the muscular arteries. α-SMA stains also evaluated the infiltration of myofibroblasts in the intima. CD45, CD3, and CD68 stains assessed the count of leukocytes, T lymphocytes, and macrophages per 10 high magnification (× 400) fields, respectively.
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4

Lymph Node Immunohistochemistry Profiling

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Lymph nodes were formalin‐fixed and paraffin‐embedded (FFPE). Paraffin sections were immunostained for CD21 (clone 1F8), CD23 (clone MHM6), Bcl‐2 (clone 124), CD10 (clone 56C6), Ki‐67 (clone Mib‐1) (Dako, Denmark), Stathmin (clone SP49; Spring Bioscience, Pleasanton, CA USA), VCAM (Clone VCAM1/843; Scytek Laboratories, Logan, UT, USA), and CXCL13 (Polyclonal Goat; R&D Systems, Minneapolis, MA, USA), using an automated immunostainer (Dako). The staining of follicular stroma and interfollicular fibroblastic stroma was graded quantitatively as previously described 20: 0, absent; 1+ focal; 2+ extensive; 3+ diffuse.
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5

Comprehensive Evaluation of Acute Myeloid Leukemia

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The Wright-Giemsa stained aspirate smears and haematoxylin and eosin (H&E) stained core bone marrow biopsies were reviewed. Immunohistochemistry studies were performed on formalin fixed, paraffin-embedded tissue with monoclonal antibodies using an automated immunostainer (Dako Omnis) using the manufacturer's instructions. Quality controls for each immunohistochemistry stain were reviewed. Flow cytometry was performed as part of routine case workup.
Standard PCR-based and cytogenetic analysis was performed at the Associated Regional and University Pathologists, Inc., ARUP Laboratories (Salt Lake City, Utah). Detection of the FLT3 TKD and IKD mutation was performed on isolated DNA using targeted fluorescent PCR primers for sequence amplification. The TKD products were cut using the EcoRV restriction enzyme and the resultant amplified ITD and TKD sequences were analyzed for base pair length on an ABI 3500xl genetic analyzer. A fragment of NPM1 exon 12 was also analyzed by targeted PCR amplification and interpreted using capillary electrophoresis. For cytogenetic studies, chromosomes were prepared from a nondiluted bone marrow aspirate collected in a heparinized syringe. The specimens were transported within 48 hours to ARUP laboratories. Each sample was cultured and suspended in metaphase. Giemsa-banded karyotyping was performed and interpreted with ISCN 2013.
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6

Quantifying Liver Fibrosis and Cell Populations

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For α‐SMA and Sox9 quantification, paraffin‐embedded mouse liver sections were stained on an automated immunostainer (Dako), using a mouse on mouse ImmPRESS HRP kit (Vector Laboratories) according to the manufacturer's instructions, using anti‐mouse α‐SMA (1:200; ASM‐1; Vector Laboratories) or anti‐rabbit Sox9 (1:500; Millipore) with a Vector ImmPRESS horseradish peroxidase rabbit secondary antibody. Ten non‐overlapping fields (×20 objective) from each section were captured using a light microscope (Carl Zeiss) with identical illumination and exposure. Digital image analysis was performed using ImageJ software for α‐SMA quantification. Sox9+ cells were counted manually. For collagen quantification, paraffin‐embedded mouse liver sections or snap‐frozen human liver sections were stained with Sirius red, using a standard protocol. Collagen values were expressed as the percentage of the total area of a section occupied by Sirius red staining.
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7

GFAP Immunohistochemistry in PDHGG

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Immunohistochemistry was carried out on formalin-fixed paraffin-embedded (FFPE) sections using an automated immunostainer (Dako Omnis). A primary antibody directed against GFAP (polyclonal, prediluited, high pH, DAKO) was applied. GFAP stained tissue slices were acquired using the Nanozoomer (Hamamatsu, RRID : SCR_022537) instrument. Slides were scanned at 40X and images were saved into.ndpi format and viewed using the NDPIv2 software (Hamamatsu). 3 random images at 20X magnification were exported from 11 PDHGG patient tissues (n=5 for H3.1 K27M; n=6 for H3.3 K27M) as.TIFF file and analyzed using the ImageJ software (RRID : SCR_003070, http://imagej.nih.gov/ij/) as described in Negm et al. (31 (link)). The mean intensity feature evaluated for each image was normalized over the number of manually counted nuclei for each image.
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8

Xenograft Tumor Analysis in NSG Mice

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We used female NSG mice (4–5 weeks old; Jackson Laboratories) for these studies. Mice were housed at the Van Andel Institute animal care facility. All animal experiments were approved by the Van Andel Institute Animal Care and Use Committee. All animal care and use protocols followed were in accordance with guidelines of the Institutional Animal Care and Use Committee. We injected mice subcutaneously in the left and right flanks with 6×106 viable control cells or RYBP/TET123 QKO cells in 0.2 mL of KSFM with Cultrex BME (R&D Systems, 3632–010–02). Mice were monitored every week for tumor formation for up to two months. Formalin-fixed, paraffin-embedded xenograft tumor tissue was sectioned and stained with hematoxylin and eosin (H&E) for histologic analysis. We performed IHC staining for CK5, p40, SNAIL and VIM at the Van Andel Institute Pathology and Biorepository Core with antibodies specified in Supplementary Table S1. IHC staining was performed on an automated immunostainer (Dako) using standard protocols and stained slides were scanned to digital files (Aperio Scanscope).
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9

Immunohistochemical Analysis of T-ALL in Pediatrics

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Formalin-fixed, paraffin-embedded tissue samples from pediatric patients with T-ALL at the time of original diagnosis were used for immunohistochemistry. Immunoperoxidase staining was performed by an automated immunostainer (DAKO, Carpinteria, CA, USA) using a standard streptavidin–biotin–peroxidase complex technique and the Ref-1 Ab (1:200; Novus Biologicals). The primary antibody was followed by HRP-conjugated goat-anti-mouse Ab, with an irrelevant IgG2 antibody (Southern Biotech) used as isotype control. Images were acquired on an Olympus microscope using an Olympus DP71 camera.
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10

Comprehensive Immunohistochemistry Profiling

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Immunohistochemistry on cell block and FFPE capsular specimens was performed using an automated immunostainer (Dako, Glostrup, Denmark) with the following primary antibodies: CD30 (clone Ber-H2), CD3 (clone F7.2.38), CD4 (clone 4B12), CD8 (clone C8/144B), CD68 (clone PG1), CD15 (clone Car-b), Granzyme B (clone GrB-7), IRF-4 (clone MUM1) (Dako), CD25 (clone 4C9, Novocastra, Newcastle Upon Tyne, UK), PAX5 (clone SP34, Thermo Scientific, Waltham, USA). Paraffin sections were pretreated using EnVision FLEX Target Retrieval Solution (Dako) and incubated with an optimal dilution of the primary antibody. The reaction was visualized with the EnVision Detection Kit (Dako) using 3–3’-diaminobenzidine chromogenic substrate. Sections were counterstained with EnVision FLEX Hematoxylin (Link) (Dako).
For every marker, positive cells were counted out of 10 non-overlapping randomly selected high-power microscopic fields (HPFs, 40×10), and the mean number of positively stained cells per HPF was recorded. The percentage of positive cells was calculated as the ratio between the mean number of stained cells per HPF and the mean number of total cells per HPF.
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