After fixation of the cells with 4% formalin in the 48-well cell culture plate and washing with PBS, the cells were permeabilized using 0,1% Triton X 100 in PBS for 10 min (200 µl per well), followed by 3 washing steps with PBS. The endogenous peroxidases were blocked with 3% H2O2 in methanol for 30 min. After this, 3 washing steps with 200 µl PBS followed and the cells were incubated for 1 h with 100 µl of a 1:1000 dilution of primary antibody (SARS-CoV-2 (2019-nCoV) Nucleocapsid Antibody, Rabbit Mab, Sinobiological Cat: 40,143-R019) in antibody diluent (REAL Antibody diluent, Agilent Technologies, Dako Cat: S202230_2) per well. Three washing steps with PBS followed and cells were treated with the secondary antibody (EnVision™ + Dual Link System HRP, Agilent Technologies, Dako Cat: K5007) for at least 30 min. After washing (PBS 3 x), the substrate (AEC substrate-Chromogen, Agilent Technologies, Dako, Cat: K346430–2, 2 drops) was dropped on the cells and incubated until viral infected cells were stained red, but not longer than 3 min. Reaction was stopped with washing in PBS (3 x) and wells were kept humid until photo documentation.
Real antibody diluent
Manufactured by Agilent Technologies
Sourced in United States
REAL Antibody Diluent is a ready-to-use protein-based buffer solution designed to dilute antibodies for immunoassays and other immunochemical applications. It helps maintain antibody stability and activity during dilution.
Automatically generated - may contain errors
Lab products found in correlation
Aec substrate chromogen, by Agilent Technologies (4 mentions)
Envision dual link system hrp, by Agilent Technologies (4 mentions)
Triton x 100, by Merck Group (2 mentions)
Eclipse ts100 microscope, by Nikon (1 mentions)
Palm microbeam microscope, by Zeiss (1 mentions)
Leica bond rxm, by Leica camera (1 mentions)
Ovation rna seq system v2 kit, by Tecan (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
S100a4 antibody, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Envision flex hrp, by Agilent Technologies (1 mentions)
Pt link, by Agilent Technologies (1 mentions)
Faramount aqueous mounting media, by Agilent Technologies (1 mentions)
Af392, by R&D Systems (1 mentions)
Clone d6b6, by Cell Signaling Technology (1 mentions)
Leica bond 3, by Agilent Technologies (1 mentions)
14 protocols using real antibody diluent
1
SARS-CoV-2 Nucleocapsid Protein Immunodetection
2
SARS-CoV-2 Nucleocapsid Protein Immunohistochemistry
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After cell fixation with 4% formalin for 20 min, the cells were transferred to an BSL-2 lab for immunohistochemistry staining. First, cells were washed with PBS and then were permeabilized with Triton X-100 (Sigma) for 10 min, followed by washing with PBS (3 times for 3 min in each washing step). Then, 3% H2O2 diluted in methanol was added for 30 min to block endogenous peroxidases, followed by the PBS washing step. Afterward the cells were incubated for 1 h with 100 µL of a 1:1000 dilution of primary antibody (SARS-CoV-2 (2019-nCoV) Nucleocapsid Antibody, Rabbit Mab, Sinobiological Cat: 40143-R019) in antibody diluent (REAL Antibody diluent, Agilent Technologies, Dako Cat: S202230_2). After a washing step, cells were treated with the secondary antibody (EnVision™ + Dual Link System HRP, Agilent Technologies, Dako Cat: K5007) for 30 min, followed again by washing. Then, the substrate (AEC substrate-Chromogen, Agilent Technologies, Dako, Cat: K346430-2) was added dropwise (2 drops) on the cells and incubated until infected cells appeared red (no longer than 3 min). The reaction was stopped by washing, and cells were kept in PBS until photo documentation.
3
Immunohistochemistry for SARS-CoV-2 Nucleocapsid Protein
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Immunohistochemistry was performed as described previously [18 (link)]. Briefly, after fixation of the cells with 4% formaldehyde, cells were permeabilized using 0,1% Triton X 100 in PBS for 10 min, and the endogen peroxidases were blocked with 3% H2O2 in methanol for 30 min. After three washing steps with PBS, the cells were incubated for 1 h with a 1:1000 dilution of primary antibody (SARS-CoV-2 (2019-nCoV) Nucleocapsid Antibody (Rabbit Mab, Sinobiological Cat: 40143-R019) in antibody diluent (REAL Antibody diluent, Agilent Technologies, Santa Clara, CA, USA, Dako Cat: S202230_2) followed by the treatment with the secondary antibody (EnVision™ + Dual Link System HRP, Agilent Technologies, Dako Cat: K5007). After washing (PBS 3x), the substrate (AEC substrate-Chromogen, Agilent Technologies, Dako, Cat: K346430-2, 2 drops) was dropped on the cells and incubated until viral infected cells were stained red. The reaction was then stopped with washing in PBS (3x) and wells were kept humid until photo documentation. For photo documentation, fourfold magnification with a Nikon Eclipse TS100 microscope (Nikon, Japan, Tokyo) was used with the JENOPTIK GRYPHAX® camera and software (Jena, Germany) were used.
4
Immunohistochemical Analysis of PALLD in FFPE Pancreatic Tissue
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After deparaffinization and rehydrating to water, FFPE tissue sections (2 μm) from the index patient were heated by microwaving for 30 minutes in pH 6 target retrieval buffer (Agilent DAKO) to unmask antibody epitopes. Nonspecific binding was blocked by protein blocking solution (5% v/v rabbit serum/antibody diluent; REAL antibody diluent, Agilent DAKO). Sections were then washed in PBS (MilliporeSigma) after each step. For PALLD IHC, sections were deparaffinized and stained with the rabbit anti-palladin (1:200 dilution, Cell Signaling Technology, 8518) antibody using a Leica Bond-RXm. The histological sections were scanned with Leica-AT2 Slidescanner, and the images were exported with Leica ImageScope Software Version 12.4.0.7018.
LCM and subsequent RNA-Seq from human pancreatic resections was performed as described previously (42 (link)). Briefly, cryosections of OCT-embedded tissue blocks from pancreatic resections collected at the Columbia Pancreas Center were transferred to PEN membrane glass slides and stained with cresyl violet acetate. LCM was performed on a PALM MicroBeam microscope (Zeiss), collecting at least 1000 cells per compartment. RNA was extracted and libraries were prepared using the Ovation RNA-Seq System V2 kit (NuGEN). Libraries were sequenced to a depth of 30 million, 100 bp, single-end reads.
LCM and subsequent RNA-Seq from human pancreatic resections was performed as described previously (42 (link)). Briefly, cryosections of OCT-embedded tissue blocks from pancreatic resections collected at the Columbia Pancreas Center were transferred to PEN membrane glass slides and stained with cresyl violet acetate. LCM was performed on a PALM MicroBeam microscope (Zeiss), collecting at least 1000 cells per compartment. RNA was extracted and libraries were prepared using the Ovation RNA-Seq System V2 kit (NuGEN). Libraries were sequenced to a depth of 30 million, 100 bp, single-end reads.
5
SARS-CoV-2 Immunohistochemical Detection
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For the immunohistochemical detection of SARS-CoV-2 in infected cells, 48-well plates were fixed for 30 min with 4% neutral-buffered formalin and were washed 3 times with PBS. Plates were incubated with 0.1 % Triton X-100 (Merck KGaA) for 10 min, washed 3x with PBS and incubated for 30 min in 3% H2O2 (Merck KGaA) dissolved in methanol (Merck KGaA, Darmstadt, Germany). After a further PBS washing step, 100 μL of the primary antibody, SARS-CoV-2 (2019-nCoV) Nucleocapsid Antibody (Rabbit monoclonal antibody (Mab); Sinobiological, China, Cat# 40,143-R019) diluted 1:1000 in REAL Antibody Diluent (Agilent Dako, Carpinteria, CA, USA, Cat# S202230−3) was added to each well. The plates were washed 3 times with PBS after 1 h incubation at RT. The Ready-to-use detection system reagent EnVision™ + Dual Link System HRP (Agilent Dako, Cat# K5007) was added for 30 min, followed by washing with PBS 3 times. AEC Substrate Chromogen (Agilent Dako, K346430−2) was applied to each well and incubated for 3 min, and the reaction was stopped by adding PBS. Wells were washed again with PBS to remove reagent and fresh PBS was added to keep the wells humid. Images were taken by light microscope (Nikon, Eclipse, TS100; Nikon Europe BV, Amsterdam, Netherlands) equipped with a JENOPTIK GRYPHAX® camera (Breitschopf, Innsbruck, Austria). SARS-CoV-2 infected cells appear red after antibody staining.
6
Immunohistochemical Evaluation of S100A4 and RAGE
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Murine cryo sections were incubated with rabbit polyclonal S100A4 antibody (Abcam, Cambridge, UK; 1:500 dilution in Real™ Antibody Diluent (Dako)). Human paraffin sections were incubated with rabbit polyclonal S100A4 antibody (Abcam; 1:700) in 10 % BSA (Sigma-Aldrich). For SMC staining rabbit polyclonal SMC α-actin antibody was used (NeoMarkers, Fermont, USA; 1:350). Anti-RAGE immunohistochemistry was performed on human and mouse paraffin sections (Abcam: 1:500). For detailed information please refer to Additional file 1 .
7
Immunohistochemical Evaluation of Bone Remodeling
8
Immunohistochemical Detection of CA IX
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Deparaffinized slides were rehydrated and immersed in PBS (10 mM, pH 7.2). Tissue epitopes were demasked using the automated water bath heating process in PT Link (Dako). Briefly, the slides were incubated in Tris-ethylenediaminetetraacetic acid (EDTA) retrieval solution (10 mM Tris, 1 mM, pH 9.0) at 98°C for 20 min. CA IX expression was detected by IHC using the in-house generated monoclonal antibody M75 against the N-terminal domain of human CA IX, as described previously (30 (link),31 (link)). Slides were incubated for 60 min at room temperature with the aforementioned primary antibody diluted to 1:100 in REAL Antibody Diluent (Dako) and immunostained using an anti-mouse/anti-rabbit immunoperoxidase polymer (EnVision FLEX/HRP; Dako) for 30 min at room temperature, according to the manufacturer's protocol. The reaction was visualized with a 3,3-diaminobenzidine substrate-chromogen solution (Dako Cytomation; Dako) for 5 min, and slides were counter-stained with hematoxylin. Clear cell renal cell carcinoma tissue was used as a positive control. As a negative control, the same tumor tissue was used, but omitting the primary antibody from the staining protocol.
9
Immunohistochemical Staining of CXCL9
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Tissue slices of 8 μm were fixed with 10% formaldehyde for 5 min, washed with TBS for 3 min and permeabilized with TBS 0.05% Tween-20 for 3 min. Endogenous enzymes were blocked 15 min with Dako dual endogenous enzyme blocker (Dako; S2003). Tissue unspecific binding was avoided by 1 h incubation with 5% bovine serum albumin, 2% powder milk, 1% human IgG in TBS 0.05% Tween-20. Primary antibody, anti-human CXCL9 goat pAb was used at 1 μg ml−1 (R&D; AF392), diluted in REAL antibody diluent (Dako; S2022) and incubated for 1 h. After three washes in TBS 0.05 % Tween-20, the secondary antibody ImmPress anti-goat-polyHRP (Vector; MP-7405) was incubated for 45 min. Slides were washed again three times and then, the substrate AEC + chromogen (Dako; K4005) was incubated for 15 min. Reaction was stopped by immersion in demineralized water. Counterstaining with hematoxilyn (solution according to Mayer; Sigma-Aldrich #51275) was done previous to mounting the slide with Faramount Aqueous Mounting Media (Dako; S3025). Images were acquired using the Mirax Digital Slide Sytem (Zeiss) and analyzed with Image J software.
10
Immunohistochemical Analysis of ALK and EGFR Mutations
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TMAs and TBB samples, sectioned at a thickness of 4 m, were stained with a rabbit anti-ALK monoclonal primary antibody (Clone D5F3, Cell Signaling Technology, Danvers, MA, USA catalog number 3633, 1:500 dilution) in Dako REAL Antibody Diluent, using an automated IHC stainer (Leica Bond-III, Dako, Trappes, France). IHC results were scored based on the staining intensity and the proportion of stained cells. The staining-intensity categories were: 0 (no staining), 1 (mild staining), 2 (moderate staining), and 3 (heavy staining). The proportion of stained cells in each category was: 0 (no cells stained), 1 (1–20% of tumor cells stained), 2 (21–50% of tumor cells stained), and 3 (>51% of tumor cells stained). If both the intensity and proportion scores were 1 or more, the sample was defined as IHC-positive. Two common EGFR mutations (exon 19, codon E746–A750 deletion and exon 21, L858R point mutation) were examined in all TMA and TBB samples using rabbit EGFR mutation-specific monoclonal antibodies [clone D6B6 (catalog number 2085) and clone 43B2 (catalog number 3197), respectively; Cell Signaling Technology].
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