For visualization of the VP1 major capsid protein of HPyV-6 in formalin-fixed paraffin-embedded tissue samples, slides were deparaffinized and rehydrated. After antigen retrieval with citrate buffer, pH 6.0 (Dako), and a wash with phosphate-buffered saline (PBS), peroxidase-blocking solution (Dako S2023) was applied for 10 minutes at room temperature. After 2 washing steps with PBS, the slides were incubated with mouse monoclonal antibodies 6V12 or 6V32, which were elicited against HPyV-6 virus-like particles using previously reported methods.20 (link) 6V12 (isotype IgG3)is specific for HPyV-6VP1, whereas 6V32 (isotype IgG1) cross-reacts with HPyV-7 VP1. Neither monoclonal antibody is reactive with the VP1 protein of MCPyV. After primary antibody binding, the slides were washed twice again with PBS and then incubated with biotinylated secondary antibody (K5003A, Dako) for 30 minutes at room temperature; after 2 more washing steps with PBS, streptavidin horseradish peroxidase (K5003B, Dako) was applied for 20 minutes at room temperature. Slides were washed twice in PBS and stained with ImmPACT NovaRED (Vector Laboratories) for 8 minutes at room temperature. After another wash in PBS, slides were counterstained with hematoxylin (Dako), rinsed in water, dehydrated, and mounted in Tissue-Tek Glas mounting medium (Sakura Finetek).
Immpact novared
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
ImmPACT NovaRED is a chromogenic substrate for detecting peroxidase activity in immunohistochemistry and other applications. It produces a red/brown precipitate at the site of the peroxidase reaction.
Automatically generated - may contain errors
Lab products found in correlation
Immpress ap reagent, by Vector Laboratories (3 mentions)
Immpact dab, by Vector Laboratories (2 mentions)
Nis elements imaging software, by Nikon (2 mentions)
Eclipse 80i microscope, by Nikon (2 mentions)
Dp71 camera, by Olympus (2 mentions)
Eclipse e400 microscope, by Nikon (2 mentions)
Ds fi1 camera, by Nikon (2 mentions)
Mouse on mouse block, by Vector Laboratories (2 mentions)
Chemidoc mp, by Bio-Rad (2 mentions)
Vectastain elite abc reagent, by Vector Laboratories (2 mentions)
S2023, by Agilent Technologies (1 mentions)
Hematoxylin, by Agilent Technologies (1 mentions)
Tissue tek glas mounting medium, by Sakura Finetek (1 mentions)
Citrate buffer ph 6, by Agilent Technologies (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
Hmb45, by Agilent Technologies (1 mentions)
Cell a imaging software, by Olympus (1 mentions)
Wash buffer, by Agilent Technologies (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
26 protocols using immpact novared
1
Immunohistochemical Detection of HPyV-6 VP1
2
Immunohistochemical Analysis of ST6GAL1 in PDAC
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Tissue microarrays containing nonmalignant and PDAC patient tissues were obtained from US Biomax Inc. (PA1001c, PA2072a, PA1921, PA1002b). Sections were IHC-stained for ST6GAL1 as reported (25 (link), 26 (link)). Briefly, sections were subjected to antigen retrieval using Antigen Unmasking Solution, Citric Acid Based (Vector Laboratories); blocked with 2.5% horse serum for 1 hour; and incubated overnight at 4°C with goat polyclonal antibody against ST6GAL1 (see Supplemental Table 3 for antibody information). Sections were incubated with ImmPRESS-HRP anti-goat IgG for 1 hour and developed using ImmPACTNovaRED or ImmPACTDAB (Vector Laboratories). Images were captured with a Nikon 80i Eclipse microscope and processed with Nikon NIS-Elements imaging software.
3
Immunohistochemical Analysis of ST6GAL1 in PDAC
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Tissue microarrays containing nonmalignant and PDAC patient tissues were obtained from US Biomax Inc. (PA1001c, PA2072a, PA1921, PA1002b). Sections were IHC-stained for ST6GAL1 as reported (25 (link), 26 (link)). Briefly, sections were subjected to antigen retrieval using Antigen Unmasking Solution, Citric Acid Based (Vector Laboratories); blocked with 2.5% horse serum for 1 hour; and incubated overnight at 4°C with goat polyclonal antibody against ST6GAL1 (see Supplemental Table 3 for antibody information). Sections were incubated with ImmPRESS-HRP anti-goat IgG for 1 hour and developed using ImmPACTNovaRED or ImmPACTDAB (Vector Laboratories). Images were captured with a Nikon 80i Eclipse microscope and processed with Nikon NIS-Elements imaging software.
4
Histopathological Analysis of Tumor Tissues
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For histopathological evaluation, hematoxylin and eosin (H&E) staining was performed on paraffin-embedded tumor tissue sections. For immunohistochemistry, sections were stained with primary antibodies overnight at 4°C. On the next day; sections were incubated for 30 minutes in ImmPRESS AP Reagent (Vector Laboratories, Burlingame, CA), followed by incubating for 2–15 minutes in ImmPACT NOVA-RED (Vector Laboratories). The following primary antibodies were purchased from Agilent (Santa Clara, CA): cancer antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), cytokeratin 20 (CK20), and Ki-67 [9 (link)]. For terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, In Situ Cell Death Detection Kit (Roche, Penzberg, Germany) was used, followed by the manufacturer’s instruction. Ki-67 positive area or TUNEL positivity in immunohistochemistry (IHC) image was measured by ImageJ software (National Institutes of Health, Bethesda, MD).
5
Teratoma Formation Assay for Pluripotent Stem Cells
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For teratoma assay about 1 × 105 or 1 × 106 mouse YFP+ ESC (pre- and post-IUT; cells after IUT were collected from 100 embryos keeping distinguishing between those isolated from AF and AM) were injected into the muscle of the hindlimb of Rag2−/−γc−/− mice straight after isolation from AF and AM digestion. After 6 weeks, mice were sacrificed and tumors collected for following analyses. Transverse sections (7–10 μm thick) of isopentan-frozen muscles were stained with Hematoxylin and eosin to evaluate the tumor tissue composition.
For immunoperoxidase staining, teratoma sections were fixed using 4% paraformaldehyde (PFA) and permeabilized with 0.1% triton X-100. βIII tubulin (1:100), α fetoprotein (1:50) and αSMA (1:100) primary antibodies were diluted in 1% BSA in PBS and incubated for 1 hour at 37 °C (for βIII tubulin) or overnight at 4 °C (for others). After peroxidase blocking, secondary antibody HRP-conjugated was incubated for 45 minutes at 37 °C. After incubation with ImmPACT NovaRED (Vector Laboratories) for 5 minutes, cytoplasm was stained with Hematoxylin (Vector Laboratories) for 9 seconds.
For immunofluorescence analysis anti–GFP 594 antibody (1:150) and anti-Laminin antibody (1:100) were used. For antibodies specification seesupplementary Table 3 .
For immunoperoxidase staining, teratoma sections were fixed using 4% paraformaldehyde (PFA) and permeabilized with 0.1% triton X-100. βIII tubulin (1:100), α fetoprotein (1:50) and αSMA (1:100) primary antibodies were diluted in 1% BSA in PBS and incubated for 1 hour at 37 °C (for βIII tubulin) or overnight at 4 °C (for others). After peroxidase blocking, secondary antibody HRP-conjugated was incubated for 45 minutes at 37 °C. After incubation with ImmPACT NovaRED (Vector Laboratories) for 5 minutes, cytoplasm was stained with Hematoxylin (Vector Laboratories) for 9 seconds.
For immunofluorescence analysis anti–GFP 594 antibody (1:150) and anti-Laminin antibody (1:100) were used. For antibodies specification see
6
Immunostaining of PAPP-A in Adipose Tissue
7
Immunohistochemistry for Melanoma Markers
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For immunohistochemistry, 5 μm tissue sections were steamed for 20 min with antigen retrieval solution and stained with primary antibodies, SOX10 (A-2, sc-365692; Santa Cruz Biotechnology, Santa Cruz, CA), HMB45 (M0634; Dako, Carpentaria, CA) and S100 (Z0311; Dako, Carpentaria, CA) overnight at 4 °C. On the next day, sections were incubated for 30 min in Imm-PRESS AP Reagent (Vector Laboratories, Burlingame, CA, USA), followed by 5 min incubation with ImmPACT NOVA-RED (Vector Laboratories).
8
Immunohistochemical and RNA Detection in Colon
9
Immunohistochemistry Staining Protocol
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Slides from step 2 were subsequently incubated with primary antibodies for 60 minutes at appropriate temperature for each type of antibody. Slides were then washed twice in wash buffer (Dako). Afterwards, incubation with secondary reagent (Envision FLEX+ mouse linker/rabbit linker, Dako) was performed for 20 minutes. For better imaging of certain antibodies, incubation with tertiary reagent (mouse anti-rabbit IgG/rabbit anti-mouse IgG, Invitrogen, Waltham, MA) was added for 30 minutes.
After washing in wash buffer (Dako) twice, slides were incubated with either anti-rabbit or anti-mouse Envision+ System horseradish peroxidase-labeled polymer for 30 minutes. For chromogenic reaction and visualization, peroxidase detection using ImmPact NovaRED (Vector Laboratories, Burlingame, CA) for 2 minutes. Slides were then washed in distilled water (DW) and wash buffer (Dako), twice each. Nuclear counterstaining using Mayer’s hematoxylin was performed, and slides were mounted and coverslipped using aqueous mount, at this point ready for whole slide scanning.
After washing in wash buffer (Dako) twice, slides were incubated with either anti-rabbit or anti-mouse Envision+ System horseradish peroxidase-labeled polymer for 30 minutes. For chromogenic reaction and visualization, peroxidase detection using ImmPact NovaRED (Vector Laboratories, Burlingame, CA) for 2 minutes. Slides were then washed in distilled water (DW) and wash buffer (Dako), twice each. Nuclear counterstaining using Mayer’s hematoxylin was performed, and slides were mounted and coverslipped using aqueous mount, at this point ready for whole slide scanning.
10
Immunohistochemical Detection of ZIKV NS2B Protein
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Paraffinized liver was cut onto slides (4 μm) and routinely deparaffinized. Slides were microwaved (GE, Model#: JES1142WD04) on high setting in Antigen Unmasking Solution (Vector Laboratories) and cooled at room temperature. Endogenous enzyme activity was blocked with BLOXALL (Vector Laboratories) and nonspecific protein binding was blocked with 10% Normal Goat Serum (Thermo Fisher). Slides were incubated with anti-ZIKV NS2B Ab (Genetex) at 4 °C for 14 hrs. Slides were incubated with ImmPRESS HRP (Vector Laboratories) secondary Ab followed by incubation with ImmPACT NovaRED (Vector Laboratories) substrate. Slides were counterstained with Modified Mayer’s Hematoxylin (Thermo Fisher) and routinely processed for mounting. Positive, negative, and rabbit IgG (Vector Laboratories) controls were included with each batch. Morphology of immuno-reactive cells were confirmed by a board-certified pathologist, and immunoreactivity was quantified by ImageDxTM (Reveal Biosciences).
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