Well-characterized anti-PD-L1 (clone 28–8; Abcam, Cambridge, MA, USA) and anti-cluster of differentiation 68 (CD68) (clone KP1; DAKO, Glostrup, Denmark) antibodies were selected. IHC was performed using an automated staining system (Leica Bond III; Leica Microsystems). The antibody dilutions were optimized to 1:100 for anti-PD-L1 and 1:400 for anti-CD68. The slides were dewaxed and rehydrated using distilled water, and were subsequently processed for PD-L1 (heat-induced antigen retrieval at pH 9.0) or CD68 (proteolytic treatment). After incubation with the primary antibodies (anti-PD-L1, 30 minutes; anti-CD68, 15 minutes), the tissue sections were rinsed, and the sections for PD-L1 staining were further incubated with EnVision FLEX+ Rabbit LINKER (DAKO) and EnVision+ HRP Labelled Polymer (DAKO). The sections for CD68 staining were incubated with the Bond Polymer Refine Detection Kit (Leica Microsystems). Staining was visualized using diaminobenzidine, and counterstaining was performed using hematoxylin. Formalin-fixed, paraffin-embedded tissue blocks of human placenta and tonsil were prepared as positive controls. The stained slides were scanned as whole-slide images using a ScanScope® Aperio CS2 slide scanner (Leica Microsystems).
Envision flex rabbit linker
Manufactured by Agilent Technologies
Sourced in United States
The EnVision FLEX+ Rabbit LINKER is a laboratory equipment product designed to facilitate the detection and visualization of proteins in biological samples. It serves as a linking agent, enabling the integration of primary antibodies raised in rabbits with the EnVision FLEX detection system.
Automatically generated - may contain errors
Lab products found in correlation
Aperio cs2 scanscope slide scanner, by Leica (1 mentions)
Bond 3, by Leica (1 mentions)
Anti pd l1 clone 28 8, by Abcam (1 mentions)
Cd68 clone kp1, by Agilent Technologies (1 mentions)
Bond polymer refine detection kit, by Leica (1 mentions)
Antibody diluent, by Agilent Technologies (1 mentions)
Envision dual link system hrp, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Envision flex hrp, by Agilent Technologies (1 mentions)
Bx45 microscope, by Olympus (1 mentions)
Diaminobenzidine dab, by Agilent Technologies (1 mentions)
Peroxidase block, by Agilent Technologies (1 mentions)
Autostainer link 48, by Agilent Technologies (1 mentions)
Autostainer plus, by Agilent Technologies (1 mentions)
Ab26405, by Abcam (1 mentions)
8 protocols using envision flex rabbit linker
1
Immunohistochemical Analysis of PD-L1 and CD68
2
Immunohistochemical Staining of BAP1 Protein
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Immunohistochemical staining for the BAP1 protein was performed using the labeled streptavidin-biotin (LSAB) staining method. In detail, sections were cut from FFPE tissue blocks at a thickness of 5 µm and mounted onto Superfrost glass slides. Sections were deparaffinized and rehydrated. Heat-mediated antigen retrieval was performed in a microwave (750 W) with citrate buffer (pH 6.0) for 10 minutes followed by cooling down for 30 minutes; application of klear dual enzyme block (E36-18; GBI Labs, Bothell, WA, USA) for 15 minutes at room temperature (RT); washing for 15 minutes (RT) with TBST (Tris buffer and Tween 20); application of protein block solution (DAKO X0909) for 10 minutes at RT; incubation with the primary antibody (rabbit anti-BAP1, Invitrogen PA5-105741, dilution 1:150 using the antibody diluent [DAKO S0809]) overnight at 4°C and 1 hour at 37°C; incubation with Envision Flex+ Rabbit Linker (DAKO K8019) for 15 minutes followed by incubation with the secondary antibody (Dako EnVision Dual Link System HRP, K4063, Lot 10101679) for 60 minutes at RT. The specimens were then stained with AEC (3-amino-9-ethylcarbazole, DAKO K3469) for 10 minutes and counterstained with Mayer's hemalum.
3
Immunohistochemical Staining of CD68 in Tissue Sections
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Five‐μm‐thick slides were deparaffinized in xylene and rehydrated in graded ethanol to distilled water. For antigen retrieval, the slides were put in citrate buffer, preheated at 60°C in a water bath, for 1 hour. After antigen retrieval, endogenous peroxidases were blocked with 3% H2O2 for 15 min. Nonspecific binding was blocked with 5% normal bovine serum in PBS for 30 min. The slides were then incubated overnight at 4°C with a rabbit anti‐CD68 primary antibody, diluted 1/2500 (specific for macrophage population, including osteoclasts cells) (Sigma‐Aldrich, St. Louis, MO, USA), followed by three washes with PBS, and incubation with secondary antibody (Envision Flex + rabbit linker; DAKO, Carpinteria, CA, USA) at room temperature for 1 hour. Then, a peroxidase polymer (Envision Flex/HRP; DAKO) was added for 30 min before revelation with a solution of DAB (DAKO). Counterstaining with Mayer's hematoxylin was finally performed. The slides were dehydrated in graded alcohol, cleared with toluene, and mounted with Eukitt mounting medium (ESBE Scientific). Histology and immunohistochemistry images were captured with an Infinity 2 camera mounted on an Olympus BX45 microscope (Olympus).
4
Immunohistochemical Detection of HNE and PRDX5 in Tissue
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4‐hydroxy‐2‐nonenal (HNE) and peroxiredoxin‐5 (PRDX5) were detected in paraffin‐embedded tissue sections (7‐μm thick). For HNE staining, sections were pretreated in citrate buffer (0.01 mol/L) in a microwave oven as described previously (Poncin et al. 2008 ). All sections were incubated with peroxidase block (DakoCytomation, Heverlee, Belgium) for 5 min at RT, washed with PBS, and incubated with PBS containing 25% nonimmune fetal bovine serum and 10% nonfat dry milk for 45 min at RT. Slides were then incubated with an anti‐HNE rabbit primary polyclonal antibody (1:300) or anti‐PRDX5 rabbit primary polyclonal antibody (1/250) (Gerard et al. 2005 ) at RT for 1 h, washed with PBS for 10 min, incubated with Envision Flex rabbit LINKER (DakoCytomation) for 15 min at RT, and then incubated with an Envision‐labeled polymer‐HRP anti‐rabbit secondary antibody (DakoCytomation) for 30 min at RT. The peroxidase reaction was visualized by incubating for 5 min with diaminobenzidine (DAB, DakoCytomation) and then counterstained with hematoxylin.
5
Immunohistochemical Analysis of MYD88, CNOT4, and EXOSC3
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To immunostain MYD88, deparaffinized sections were exposed to EnVision FLEX Target Retrieval Solution High pH (50×) (Dako) for 20 min at 97°C for activation, and Autostainer Link 48 (Dako) was used. IHC was performed using rabbit polyclonal anti‐MyD88 antibodies (1:50 dilution; HFL‐296, Santa Cruz Biotechnology, Inc). The sections were sensitized using EnVision FLEX+ Rabbit (LINKER) (Dako) for 15 min.
For immunohistochemical analysis of CNOT4 and EXOSC3, anti‐CNOT4 (ab110013, Abcam) and anti‐EXOSC3 (ab154400, Abcam) antibodies were used, and IHC was performed according to the standard protocol. The staining intensity was visually scored as follows: − (negative), 1+ (weak), 2+ (moderate), and 3+ (strong). The immunostaining of endothelial cells was evaluated as an internal control.
For immunohistochemical analysis of CNOT4 and EXOSC3, anti‐CNOT4 (ab110013, Abcam) and anti‐EXOSC3 (ab154400, Abcam) antibodies were used, and IHC was performed according to the standard protocol. The staining intensity was visually scored as follows: − (negative), 1+ (weak), 2+ (moderate), and 3+ (strong). The immunostaining of endothelial cells was evaluated as an internal control.
6
Immunohistochemistry of Human Duodenum
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Sections of duodenum from humans were prepared and mounted onto Superfrost Plus slides for immunohistochemistry (IHC). IHC for mitotic marker Ki-67 was performed using an automated machine (Autostainer Plus, Dako, Denmark) using Dako reagents and following standard protocols supplied by the manufacturer (FLEX Plus Detection System, Dako). Briefly, sections were deparaffinised in histolene, rehydrated through graded ethanols and treated in Dako PT Link (pretreated module) with preheated (65°C) FLEX Tris/EDTA at pH 9 retrieval solution, until solution reached 97°C where it stayed for 20 min. It was then cooled back down to 65°C and dipped into the Dako wash buffer. Non-specific staining was blocked by 100 µL FLEX Peroxidase Block and 100 µL serum-free protein block. Primary antibody (ab16667) was diluted in Dako antibody diluent and remained for 60 min, followed by application of 100 µL of the EnVision FLEX+Rabbit LINKER (#K8019, Dako) and 150 µL of diaminobenzidine (DAB) for 10 min. Sections were counterstained with Lillie-Mayer’s haematoxylin, dehydrated, cleared and coverslipped. Archived colon tumour sections were used as the positive control. Omission of the primary antibody was used as the negative control.
7
Immunohistochemical Detection of LASV Nucleoprotein
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For immunohistochemical analysis, antigen was retrieved from formalin-embedded tissues with EnVision FLEX Target Solution (Agilent Technologies, Cat# GV80411-2) at high PH (Code k8004), at 97 °C for 20 min. Slides were then blocked for 10 min with Dako’s serum-free protein blocking reagent (Agilent Technologies, Cat# X090930-2). Next, the slides were stained with a LASV nucleoprotein rabbit polyclonal antibody (GeneTex, Cat# GTX134883) at 1:4000 for 30 min at room temperature. The slides were then treated with Envision Flex Rabbit Linker at high pH for 15 min (Agilent Technologies, Cat# K800921-2). Next, EnVision HRP was put on the slides for 15 min, followed by EnVision FLEX DAB+ Substrate Chromagen for 5 min (Agilent Technologies, Cat# GV82511-2). Finally, slides were counterstained with Mayer’s Hematoxylin.
8
Immunohistochemical Characterization of Pancreatic Tissue
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Serial sections from archival pancreatic tissue were subjected to MAFA immunohistochemistry as well as insulin and glucagon staining. The result was evaluated and compared to that of normal human pancreas by an experienced pathologist. To achieve comparability, MAFA immunohistochemistry was performed using the same MAFA antibody (Anti-MafA antibody ab26405, Abcam, Cambridge, UK, RRID: AB_776146; dilution: 1:2000) as Iacovazzo et al. [12 (link)] following the manufacturer’s instructions. After deparaffinization, hydration, and heat-induced epitope retrieval, incubation steps were carried out in an Autostainer 480-A (Thermo Fisher Scientific, Dublin, Ireland) using the Dako EnVision™ FLEX HRP/Dab (Agilent, Santa Clara, CA, USA) detection system with signal amplification by EnVision™ FLEX + Rabbit (LINKER) (Agilent, Santa Clara, CA, USA).
Standard staining/immunohistochemisty protocols of the Institute of Pathology (University Medical Center Mainz) were used to detect the neuroendocrine/proliferation markers chromogranin, synaptophysin, insulin, glucagon, and KI-67 in formalin-fixed, paraffin-embedded pancreatic tissue sections from patients IV3 and IV4 following resection.
Standard staining/immunohistochemisty protocols of the Institute of Pathology (University Medical Center Mainz) were used to detect the neuroendocrine/proliferation markers chromogranin, synaptophysin, insulin, glucagon, and KI-67 in formalin-fixed, paraffin-embedded pancreatic tissue sections from patients IV3 and IV4 following resection.
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