Tissue sections were processed for IHC as previously described (32 (link), 35 (link)). In brief, cortical areas of all 13 GME cases and four unaffected dogs were evaluated with antibodies to CD3 (T cells) (Agilent (DAKO), M7254, Ms mAb), Multiple Myeloma 1 (MUM1/IRF4) (plasma cells) (Dako, M7259, Ms mAb), CD79b (B cells) (CST, #96024, Rb mAb), Iba1 (macrophages/microglia) (WAKO, 019-19741, Rb pAb), myelin-oligodendrocyte glycoprotein (MOG) (myelin) (CST, #96457, Rb mAb). All of these stainings were performed using a LEICA Bond RXm automated IHC stainer according to manufacturer’s recommendations. Isotype control antibodies were used as negative controls. Positive controls were normal canine brain tissue and lymph node. All GME and control IHC slides stained immunohistochemically were scanned at 20x using a Leica Aperio AT2 slide scanner (Leica Biosystems, Inc., Buffalo Grove, IL) and image acquisition was performed with ImageScope (Leica Biosystems, Inc., Buffalo Grove, IL). For the quantification of the MOG staining to assess demyelination, image analysis was performed using a positive pixel count algorithm using ImageScope software. A single algorithm was developed using appropriate thresholds for the staining intensity, and was applied to specific annotated regions on each slide.
M7254
Manufactured by Agilent Technologies
Sourced in United States, United Kingdom
The M7254 is a precision digital multimeter produced by Agilent Technologies. It is a versatile instrument designed for accurate measurement of voltage, current, and resistance in a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Leica aperio at2 slide scanner, by Leica Biosystems (1 mentions)
Bond rxm, by Leica camera (1 mentions)
Imagescope, by Leica Biosystems (1 mentions)
Rb 9013 p, by Thermo Fisher Scientific (1 mentions)
K3468, by Agilent Technologies (1 mentions)
E0353, by Agilent Technologies (1 mentions)
P0397, by Agilent Technologies (1 mentions)
Ab24070, by Abcam (1 mentions)
Glycol methacrylate, by Polysciences (1 mentions)
Envision flex kit, by Agilent Technologies (1 mentions)
M0752, by Agilent Technologies (1 mentions)
M7103, by Abcam (1 mentions)
M0821, by Agilent Technologies (1 mentions)
Ab52587, by Abcam (1 mentions)
Sc 373799, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Luminata western hrp substrate, by Merck Group (1 mentions)
Hyperpage prestained protein marker, by Meridian Bioscience (1 mentions)
Actin, by Merck Group (1 mentions)
Immobilon, by Merck Group (1 mentions)
7 protocols using m7254
1
Immunohistochemistry of Canine GME Brains
2
Feline Lymphoma Histopathological Analysis
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Six of the cats (DR, R1, R4, P1, P2 and P5; Table 1 ) had to be euthanized for humane reasons and underwent necropsy and histopathological examination. Tissue sections from the lymphoma were tested to identify B and T cells using a CD3 T-cell marker (M7254, DAKO) and the B-cell marker for CD79 (M7051, DAKO; RB-90-13-P, Labvision, Thermo Fisher Scientific, Fremont, USA).
3
Immunohistochemical Analysis of CD3 and FoxP3
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Formalin-fixed, paraffin-embedded NT and TT sections were stained for CD3 and FoxP3 markers by immunohistochemistry. Tissue sections were de-paraffinized in xylene and rehydrated with decreasing concentrations of ethanol. Antigen retrieval was performed at 90°C for 5 min using citrate buffer (pH 6.0). After serial blocking with 3% hydrogen peroxide (in methanol) and protein block (1% bovine serum albumin, 0.05% Tween 20 in PBS), tissue sections were incubated overnight at 4°C with primary monoclonal antibodies against CD3 (M7254, Dako) and FoxP3 (14-4777-82, eBioscience). The sections were then incubated at room temperature for one hour with biotinylated immunoglobulin (E0353, Dako) and another one hour with peroxidase-conjugated streptavidin (P0397, Dako) followed by the addition of DAB+ substrate chromogen (K3468, Dako) for color development. Sections were then counterstained by hematoxylin solution.
4
Immunohistochemical Profiling of Airway Biopsies
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Mucosal biopsy specimens were processed into glycol methacrylate (Polysciences, Northampton, United Kingdom). Two-micrometer sections were stained with antibodies: DP2/CRTH2 (rabbit polyclonal against sequence CAASPQTGPLNRALSSTSS, 1 μg/mL; AstraZeneca, London, United Kingdom) with staining confirmed by an alternative antibody to DP2/CRTH2 OPA1-15328 (5 μg/mL; Thermo Fisher Scientific, Leicestershire, United Kingdom), mast cell tryptase (IR640; Dako, Cambridge, United Kingdom), CD3 (M7254, 5 μg/mL; Dako), major basic protein (MON6008, 1.3 μg/mL; Monosan, Newmarket, United Kingdom), neutrophil elastase (M0752, 0.02 μg/mL; Dako), CD4 (M7310, 5 μg/mL), CD8 (M7103, 1 μg/mL), MUC5AC (Ab24070, 1 μg/mL; Abcam, Cambridge, United Kingdom), pancytokeratin (M0821, 1 μg/mL; Dako), involucrin (Ab68, 0.75 μg/mL; Abcam), or isotype controls (Dako). The EnVision FLEX kit (Dako) was used. Colocalization was undertaken with sequential sections, as described previously.21 (link) Positively stained nucleated cells were enumerated per square millimeter of submucosal area, per 10 mm2 of total epithelial area, or per millimeter of ALI culture length by a blinded observer. Grading criteria were derived for histology of biopsy specimens and area of involucrin-positive staining. Grading was carried out on 2 separate occasions by a blinded observer.
5
Western Blotting of Immune Proteins
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Human specimen samples for western blotting were snap-frozen in liquid nitrogen and stored at -80ºC. Human protein extracts were obtained using ice-cold lysis buffer as described [19 (link)]. Protein content of lysates was determined by the bicinchoninic acid (BCA) protein assay (Pierce) using bovine serum albumin as standard. Protein lysates (15 µg/lane) were resolved using sodium dodecyl sulphate–polyacrilamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Immobilon, Merck-Millipore; Burlington MA, USA, IPVH00010). Blots were blocked with 5% non-fat dry milk in TBST buffer (100 mM sodium chloride, 10 mM Tris–HCl pH 7.5, 0.05% Tween-20) at room temperature for 1 h. Membranes were then incubated overnight with mouse monoclonal antibodies against: PD-1 (ab52587, Abcam, Cambridge, UK), CD69 (sc-373799, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), NOR-1 (H00008013-M06, Abnova, Taipei, Taiwan) or CD3 (M7254, Dako). Appropriate horseradish peroxidase-conjugated secondary antibodies (Dako) and the Luminata™ Western HRP Substrate (Immobilon, Merck-Millipore) were used to detect bound antibodies. The size of detected proteins was estimated using protein molecular mass standards (Hyperpage Prestained Protein Marker; Bioline, Paris, France). ß-actin (A5441, Sigma-Aldrich) was used to verify equal loading of protein on each lane.
6
Immunostaining of Human Pancreatic Islets
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Human pancreas sections were deparaffinized, followed by acidic-pH heat–mediated antigen retrieval. Cultures of single pancreatic islet cells were fixed with 4% paraformaldehyde. Samples were blocked and permeabilized in PBS with 0.3% Triton X-100 and 10% goat or donkey serum. Primary antibodies CHOP (1:100; sc-575, Santa Cruz Biotechnology), GAD6 mouse monoclonal antibody (mAb) against C-terminus of GAD65 (31 (link)) (1:1,000); N-GAD65 mouse mAb against the N-terminus of GAD65 (32 (link)) (1:300), giantin (1:1,000; ab24586, Abcam); insulin (1:10,000; 4011-01, Linco); insulin (1:2,000; ab14042, Abcam), and CD3 (1:30; M7254, Dako) were incubated overnight at 4°C in PBS with 0.3% Triton X-100 and 1% goat or donkey serum. Alexa Fluor conjugated secondary antibodies (Molecular Probes) were incubated at 1:200 dilution in PBS with 0.3% Triton X-100 for 30 min at room temperature.
7
Histological Analysis of Decalcified Bone
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For Gram staining, decalcified bone samples were treated with 10 % (w/v) crystal violet (Sigma Aldrich, MO, USA) followed by Lugol's solution(Sigma Aldrich). Differentiation was performed with acetone, following by staining with 10 % (v/v) Ziehl-Neelsen's carbol fuchsin (Merck Millipore, MA, USA). Picric acid was used as a counterstain (VWR). To localise Gram-positive bacteria, a modified Gram stain was used in which samples were counterstained with 0.05 % (w/v) light green SF (Sigma Aldrich) after the www.ecmjournal.org differentiation step. To identify mycobacteria, an acid-fast bacteria stain kit was used according to the manufacturer's instructions (Dako). Tartrate-resistant acid phosphatase (TRAP) activity was visualised to demonstrate the presence of osteoclasts as described before (Croes et al., 2017a) . Immunohistochemical staining was performed as previously described in detail (Croes et al., 2017b) . A mouse-anti-human calprotectin antibody (5 μg/mL, MCA874A488, clone MAC387, Bio-rad) was used to detect activated macrophages and neutrophils. For detection of T lymphocytes, a mouse-anti-human CD3 antibody (0.7 mg/mL, M7254, clone F7.2.38, Dako) was used. For each sample, the total number positively-stained cells in one cross section was counted.
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