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Plus amplification diluent

Manufactured by PerkinElmer
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1X Plus Amplification Diluent is a solution designed for use in molecular biology applications that require sample dilution. It is a ready-to-use buffer that maintains the integrity of nucleic acid samples.

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Lab products found in correlation

Histofine simple stain kit, by Nichirei Biosciences (3 mentions) Malinol, by Muto Pure Chemicals (3 mentions) Vectastain elite abc kit, by Vector Laboratories (2 mentions) Biotinyl tyramide stock solution, by PerkinElmer (2 mentions) Tsa biotin system, by PerkinElmer (2 mentions) Tsa biotin system kit, by PerkinElmer (1 mentions) Psd95, by Cell Signaling Technology (1 mentions) Calbindin d28k, by Santa Cruz Biotechnology (1 mentions) Dab 3 3 diaminobenzidine, by Merck Group (1 mentions) Biotinyl tyramide, by PerkinElmer (1 mentions) Vectastain elite abc reagent, by Vector Laboratories (1 mentions) Ab5026, by Merck Group (1 mentions) Alexa fluor 594 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions) Prolong gold, by Thermo Fisher Scientific (1 mentions) Background reducing components, by Agilent Technologies (1 mentions)

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Amplification diluent (4 citations)

5 protocols using plus amplification diluent

1

Immunohistochemical Detection of TH and MEnk

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The sections were incubated for 18 h in 3% BSA-PBS containing a rabbit polyclonal antibody against TH (1:200,000; Sato et al., 2008 (link)) or [Met]-enkephalin (MEnk; 1:500,000; Millipore, St. Louis, MO, USA; Goto et al., 2015 (link)). They were then processed for a hybrid IHC protocol that implements aspects of both the polymer-staining and avidin-biotin-complex (ABC) methods combining with the biotin-TSA system, as we previously reported (Goto et al., 2015 (link)). Briefly, the sections were incubated for 30 min in the polymer-staining solution (Histofine Simple Stain Kit; Nichirei). After several rinses in PBS, they were incubated for 30 min with biotinyl TSA solution prepared from a TSA Biotin System kit (Perkin Elmer) with 1× Plus Amplification Diluent (Perkin Elmer). After several rinses in PBS, they were incubated for 30 min with the ABC reagent prepared from a Vectastain Elite ABC kit (Vector). After several rinses in PBS, the sections were immersed for 10 min in 0.05 M Tris-HCl (pH 7.4) containing 0.05% diaminobenzidine (DAB; Merck, Darmstadt, Germany) and 0.01% H2O2. After dehydration, the sections stained with DAB were cover-slipped with Malinol (Muto Pure Chemicals, Tokyo, Japan).
Morigaki R, & Goto S. (2016). Putaminal Mosaic Visualized by Tyrosine Hydroxylase Immunohistochemistry in the Human Neostriatum. Frontiers in Neuroanatomy, 10, 34.
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2

Immunohistochemical Visualization of PSD-95 and Calbindin-D28K

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The sections were incubated with a rabbit polyclonal antibody against PSD-95 (1:5,000; Cell Signaling) or a goat polyclonal antibody against Calbindin-D28K (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 18 h in PBS containing 3% BSA. After several rinses in PBS, the sections were incubated with the polymer-staining reagent (Histofine Simple Stain Kit; Nichirei) for 30 min. After several rinses in PBS, they were processed for TSA using the TSA Biotin System (Perkin Elmer). Sections were then incubated in the biotinyl tyramide amplification reagent. A working solution was prepared by diluting the Biotinyl Tyramide Stock Solution (Perkin Elmer) 1:50 using 1× Plus Amplification Diluent (Perkin Elmer) for 30 min. After several rinses in PBS, the sections were incubated for 30 min with the avidin-biotin-peroxidase complex (ABC) reagent from a Vectastain Elite ABC kit (Vector). The bound peroxidase was visualized by incubating the sections with a solution containing 0.05% 3,3′-diaminobenzidine (DAB; Merck, Darmstadt, Germany) and 0.01% H2O2 in 0.05 M Tris-HCl (pH 7.4) for 10 min. The immunostained sections were dehydrated and cover-slipped with Malinol (Muto Pure Chemicals, Tokyo, Japan).
Morigaki R, & Goto S. (2015). Postsynaptic Density Protein 95 in the Striosome and Matrix Compartments of the Human Neostriatum. Frontiers in Neuroanatomy, 9, 154.
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3

Immunostaining Protocol for Methionine-Enkephalin

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The sections were incubated with rabbit polyclonal antibody against MEnk (AB5026 from Millipore; 1:50,000, 1:500,000, or 1:5,000,000) or without the anti-MEnk antibody for 18 h in PBS-BSA. After several rinses in PBS, the sections were incubated with the polymer staining reagent (Histofine Simple Stain Kit; Nichirei, Tokyo, Japan) for 30 min. After several rinses in PBS, they were processed for tyramide signal amplification (TSA) using the TSA Biotin System (Perkin Elmer, Boston, MA, USA). The sections were incubated in Biotinyl Tyramide (amplification reagent) Working Solution that was made by diluting Biotinyl Tyramide Stock Solution (Perkin Elmer) 1:50 using 1X Plus Amplification Diluent (Perkin Elmer, FP1135) for 30 min. After several rinses in PBS, the sections were incubated with the Vectastain Elite ABC reagent (Vector) for 30 min in PBS. After several rinses in PBS, the bound peroxidase was visualized by incubating the sections with a solution containing 0.05% DAB and 0.01% H2O2 in 0.05M Tris-HCl (pH 7.4) for 10 min. After several rinses in water, the immunostained sections were dehydrated and cover-slipped with Malinol (Muto Pure Chemicals). Overview protocol for the PBTA-DAB staining is shown in Figure 1 (left).
Goto S., Morigaki R., Okita S., Nagahiro S, & Kaji R. (2015). Development of a highly sensitive immunohistochemical method to detect neurochemical molecules in formalin-fixed and paraffin-embedded tissues from autopsied human brains. Frontiers in Neuroanatomy, 9, 22.
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4

Immunohistochemical Protocol for Retinal Labeling

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Immunohistochemical protocols were as reported previously (Estevez et al., 2012) (link) and described here: Retinas were fixed for 1 h in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PBS), then rinsed in 0.1 M PBS (6 × 10 min). Retinas were soaked overnight at 4°C in a PBS solution of 2% Triton X-100 and 5% donkey serum, then incubated for two days at 4°C in primary antibody, rinsed in PBS (6 × 10 min), then incubated for 2-4 hrs at 4°C in secondary antibody and finally washed in PBS (3 × 15 min). The primary antibodies were goat polyclonal anti-choline acetyltransferase (1:200; ChAT; Millipore, Temecula, CA) and rabbit polyclonal anti-melanopsin (1:10,000; ATS-Advanced Targeting Systems, San Diego, CA). Secondary antibodies were Alexa Fluor 594 or 647 donkey anti-goat IgG and Alexa Fluor donkey anti-rabbit 594 (1:200; Invitrogen-Molecular Probes, Eugene, OR). In some cases, the sensitivity of melanopsin immunodetection was increased by tyramide signal amplification with horseradish peroxidase (HRP)-tagged goat anti-rabbit IgG and Alexa Fluor 594 tyramide (TSA-15, Molecular Probes, Eugene, OR), using the manufacturer’s protocol exactly with the exception of PerkinElmer 1X Plus Amplification Diluent which replaced the diluent included in the kit. Retinas were mounted on glass slides and coverslipped using Aqua-Mount or ProLong Gold (Invitrogen, Carlsbad, CA)
Stabio M.E., Sabbah S., Quattrochi L., Ilardi M.C., Fogerson P.M., Leyrer M., Renna J.M., Kim M.T., Kim I., Schiel M., Briggman K.L, & Berson D.M. (2017). The M5 Cell: A Color-Opponent Intrinsically Photosensitive Retinal Ganglion Cell. Neuron, 97(1), 150-163.e4.
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5

Multiparametric Immunofluorescence Profiling

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All steps were performed at room temperature. Serial 2-μm sections of each specimen were labeled with a first marker and subsequently with 4 other markers (Table 1). The “macrophage panel” includes CD68 (pan-macrophage), CD86 (M1), CD105 (activated macrophages), and also CD163 and CD206 both (M2). The “neutrophil panel” includes CD15 (neutrophils), histone H3, MPO, NE, and CD68. The “lymphocyte panel” includes CD3 (pan-T-lymphocyte), CD4 (T-helper cell), CD8 (cytotoxic T cell), CD20 (pan-B-lymphocyte), and CD68 (Supplementary Figure 1 in Supplementary Material 1).
The order of the fluorophores or fluorescent dyes was always kept the same for all panels; OpalTM 480 was used first, followed by OpalTM 520, OpalTM 570, OpalTM 650, and finally OpalTM 780. All antibodies used were monoclonal and diluted with antibody diluent (with Background Reducing Components, Dako, Germany). Secondary antibodies were applied with ImmPRESSTM HRP (peroxidase) Polymer Detection Kit (Vector, Laboratories, US). Fluorochromes were diluted with 1x Plus Amplification Diluent (PerkinElmer, US).
Klinge U., Dievernich A, & Stegmaier J. (2022). Quantitative Characterization of Macrophage, Lymphocyte, and Neutrophil Subtypes Within the Foreign Body Granuloma of Human Mesh Explants by 5-Marker Multiplex Fluorescence Microscopy. Frontiers in Medicine, 9, 777439.
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