Fresh graft tissue samples were fixed in 10% phosphate buffered formalin (Thermo Fisher Scientific, Waltham, MA) and embedded in paraffin blocks. Five-micron sections were deparaffinized, and antigen retrieval was performed using a decloaker as previously described.7 (link) The slides were rinsed and cooled in phosphate buffered saline. Endogenous peroxidase activity was blocked by incubation for 5 minutes with Dual Endogenous Enzyme-Blocking Reagent (Agilent, Santa Clara, CA, USA). Successive sections were then stained to assess for the expression of CD20 (Clone L26; Agilent). For immunofluorescence, the following antibodies were used: CD27 (clone EPR8569; Abcam, Cambridge, MA) and CD20 (Clone L26; Agilent). Donkey antirabbit (AF488; Life Technologies, Carlsbad, CA) and donkey anti-mouse (NL557; R & D Systems, Minneapolis, MN) were used as secondary antibodies.
Dual endogenous enzyme blocking reagent
Manufactured by Agilent Technologies
Sourced in United States, Denmark
The Dual Endogenous Enzyme-Blocking Reagent is a laboratory equipment product designed to inhibit the activity of endogenous enzymes in biological samples. It functions to prevent interference from these enzymes during various analytical and experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Hpa001906, by Merck Group (3 mentions)
3 3 diaminobenzidine (dab), by Merck Group (3 mentions)
Vectastain abc hrp kit, by Vector Laboratories (2 mentions)
Dab 3 3 diaminobenzidine hrp substrate, by Vector Laboratories (2 mentions)
Normal goat serum block, by Santa Cruz Biotechnology (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
Hpa008736, by Merck Group (2 mentions)
Citrate buffer ph 6, by Vector Laboratories (2 mentions)
Mayer s hematoxylin, by Agilent Technologies (2 mentions)
Powervision poly hrp ihc detection system protocol, by Leica (2 mentions)
Dab chromogen, by Merck Group (2 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Aec substrate chromogen, by Agilent Technologies (2 mentions)
Buffered formalin phosphate, by Thermo Fisher Scientific (1 mentions)
Cd20 clone l26, by Agilent Technologies (1 mentions)
St4020 small linear stainer, by Leica (1 mentions)
Antibody diluent, by Agilent Technologies (1 mentions)
Bz x710 inverted fluorescence microscope, by Keyence (1 mentions)
Decloaking chamber dc2012, by Biocare Medical (1 mentions)
Liquid dab substrate chromogen system, by Agilent Technologies (1 mentions)
19 protocols using dual endogenous enzyme blocking reagent
1
Immunohistochemical Analysis of CD20 and CD27 in Tissue Samples
2
Immunohistochemical Staining Protocol
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Sections were cut to 4 μm thickness and placed on frosted histology glass slides (Thermo Fisher). H&E stained sections were obtained from each FFPE block. Deparaffinization, rehydration, and HIER were performed on an ST4020 small linear stainer (Leica) as described above. Nonspecific binding was blocked for 1 h at room temperature using 100 μL of serum-free protein block (Agilent). Antibodies were diluted in 100 μL antibody diluent (Agilent), and sections were stained overnight in a sealed humidity chamber at 4°C on a shaker. After staining, slides were washed for 10 min in 1x TBS IHC wash buffer with Tween® 20 (Cell Marque), and specimens were covered with dual endogenous enzyme-blocking reagent (Agilent) for 5-10 min at room temperature, followed by washing for 10 min. Bound antibodies were then visualized using the HRP/liquid DAB+ substrate chromogen system (Agilent) according to the manufacturer’s instructions. Sections were counterstained with hematoxylin, followed by dehydration, mounting, and imaging in brightfield mode on a BZ-X710 inverted fluorescence microscope (Keyence).
3
FFPE Tissue Immunohistochemical Staining
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All tissue samples were FFPE, sectioned (3–4 μm-thick) and dried for 1 h at 65 °C. Next, tissue samples were subjected to deparaffinization, rehydration and heat-induced epitope retrieval using a Biocare Decloaking Chamber (DC2012) at 110 °C for 6 min in the corresponding unmasking solution (Supplementary Table 5 ). The endogenous peroxidase was blocked with dual endogenous enzyme-blocking reagent (#S2003, Agilent) for 10 min. Reagents were incubated at room temperature in a humidified slide chamber. Detailed information about the antibodies, dilutions and conditions used for the immunohistochemical stainings are specified in Supplementary Table 5 . All the stainings were counterstained with Haematoxylin.
4
Immunohistochemical Analysis of EGFR in FFPE Tissues
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Formaldehyde-fixed paraffin-embedded (FFPE) tissues were prepared using standard methods as described in77 (link). Morphologic analysis of tumor tissues was performed on hematoxylin- and eosin-stained sections. For antibody staining, sections were blocked (Dual Endogenous Enzyme Blocking Reagent, Dako, S200389-2) in 1% (w/v) BSA for 60 min at RT, incubated with primary antibodies (Anti-EGFR, D38B1, CST 4267; RRID: AB_2246311) at 4 °C overnight and secondary antibody (2 µg/mL Anti-Rabbit Ig-HRP, Dako P044801 in Tris Borate Saline - TBS) for 60 min at RT. Samples were developed (using Liquid DAB+ Substrate Chromogen System, Dako, K3467) and counterstained with hematoxylin before mounting. Slides were scanned using a Nanozoomer (Hamamatsu, Japan) with images being analyzed and processed by ImageJ.
5
Immunohistochemical Detection of CD276
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Formalin-fixed paraffin embedded (FFPE) sections were deparaffinized, treated with Dual Endogenous Enzyme-Blocking Reagent (Dako) followed by the Biotin blocking system (Dako) and blocked with 1% blocking reagent (Roche) in TBS (100 mM Tris (pH 7.5), 150 mM NaCl + 1% Triton X 100). Sections were incubated with goat anti-human CD276 (R&D catalogue # AF1027) for 2 hr at room temperature followed by signal amplification using a Vectastain ABC HRP Kit (Vector Laboratories). The goat anti-human CD276 antibody from R&D used for IHC staining of human PDX tumor tissues was also found to react specifically with murine CD276 in tumor vessels of CD276 WT but not KO mice14 (link).
6
Ki-67 Immunohistochemical Staining of Tumor Tissue
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For immunohistochemical staining, a 5‐μm section of the tumor tissue was deparaffinized, rehydrated, and subjected to antigen retrieval (Trilogy, Cell Marque, Hot Springs, AR). After cooling for 20 min at room temperature, the retrieval solution was decanted and the sample washed three times at room temperature using a phosphate‐buffered saline solution. After tissue blocking using a commercial blocking solution (Dual Endogenous Enzyme‐Blocking Reagent, Dakocytomation), the primary antibodies for Ki‐67 (1:100, Dako; MIB‐1) were added, and the specimen was incubated at 4°C overnight. Ki‐67 staining was then performed using the Ventana Autostainer (iVIEW DAB Detection Kit, Ventana Medical Systems, Tuscon, AZ) before all sections were counterstained with hemalum. Two observers reviewed each slide and performed Ki‐67 scoring by determining the percentage of positive nuclei from regions of maximal nuclear staining after counting more than 600 cells at ×400 magnification.
7
Optimized Immunohistochemical Staining for Osteosarcoma
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Immune staining was optimized for proper antigen retrieval and antibody concentrations using immune tissue for positive controls and brain tissue for negative controls. Osteosarcoma tumor slides were baked at 60 °C for one hour and antigen retrieval was performed using either tri-citrate buffer, pH 6.0 or tris-EDTA buffer pH 8.0 at 100 °C for 20 minutes. Slides were de-paraffinized using xylene, rehydrated along an ethanol gradient, and blocked using dual endogenous enzyme-blocking reagent (Dako) and a subsequent normal goat serum block (Santa Cruz). Slides were incubated overnight at 4 °C in primary antibody followed by a one hour room-temperature incubation with secondary antibody. Antibody concentrations and information can be found in Supplementary Table 1 . Antigen expression was detected by addition of VECTASTAIN Elite ABC Kit (Vector Laboratories) and DAB (3,3-diaminobenzidine) HRP substrate (Vector Laboratories). Slides were counterstained with hematoxylin (Harris). Human placenta was used as positive control of PD-L1 expression. A full list of antibodies and their positive controls for all antibodies can be found in Supplementary Table 1 .
8
Immunohistochemical Analysis of ATRX and DAXX
9
Immunohistochemical Analysis of ATRX and DAXX
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Immunohistochemistry (IHC) was done to confirm the effect of the mutations on the ATRX protein in the FFPE tumor tissue. Slides were deparaffinized in xylene and rehydrated in a graded ethanol series. Heat-induced antigen retrieval was performed in a steamer using citrate buffer (pH 6.0) (Vector Laboratories) for 30 min followed by 10 min of cooling. Endogenous peroxidase was blocked for 10 min with dual endogenous enzyme-blocking reagent (Dako). Serial sections were then incubated with primary antibody; anti-ATRX (1:400) (Sigma-Aldrich; HPA001906) and anti-DAXX (1:75) (Sigma-Aldrich; HPA008736) for 1 hour at room temperature. The sections were then incubated for 30 min with secondary antibody (Leica Microsystems) followed by detection with 3,3′-Diaminobenzidine (Sigma-Aldrich) for 8 min. Sections were washed with phosphate-buffered saline with 0.1% Tween-20. Finally, sections were counterstained with hematoxylin, subsequently rehydrated in a graded ethanol series and mounted. Only nuclear labeling of either protein in tumor cells was considered positive. Endothelial cells on each section served as internal controls.
10
Insulin and Glucagon Immunohistochemistry
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Antigen retrieval was performed on paraffin-embedded sections with Retrievagen A Solution (BD Biosciences), and endogenous biotin was blocked by Dual Endogenous Enzyme Blocking Reagent (Dako). Guinea pig antibody to insulin (Dako) or rabbit antibody to glucagon (Abcam) was added and detected with the EnVision Dual Link Kit (Dako) followed by staining with DAB Solution (Dako). Samples were counterstained with hematoxylin. A veterinary pathologist scored histopathological changes by blinded scoring of sections.
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