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Fp1487001kt

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FP1487001KT is a laboratory equipment product. It serves a core function in scientific research and analysis processes. A detailed description is not available while maintaining an unbiased and factual approach.

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Lab products found in correlation

Fp1488001kt, by Akoya Biosciences (5 mentions) Fp1497001kt, by Akoya Biosciences (4 mentions) Fp1495001kt, by Akoya Biosciences (3 mentions) Rnascope fluorescent v2 kit, by Bio-Techne (2 mentions) Multiplex fluorescence v 2 kit, by Advanced Cell Diagnostics (2 mentions) A11122, by Thermo Fisher Scientific (1 mentions) Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions) Nef812001ea, by PerkinElmer (1 mentions) Bond er2, by Leica (1 mentions) Goat anti rabbit poly hrp secondary antibody, by Thermo Fisher Scientific (1 mentions) Antibody diluent block ard1001, by PerkinElmer (1 mentions) Bond rx, by Leica (1 mentions) Prolong gold antifade reagent mounting medium, by Thermo Fisher Scientific (1 mentions) Paraformaldehyde (pfa), by Solarbio (1 mentions) Cm3050s cryostat, by Leica (1 mentions) A1rsi confocal microscope, by Nikon (1 mentions) Eclipse ti2 microscope, by Nikon (1 mentions) Rnascope probe, by Advanced Cell Diagnostics (1 mentions) D1306, by Thermo Fisher Scientific (1 mentions) Prolong diamond, by Thermo Fisher Scientific (1 mentions)

14 protocols using fp1487001kt

1

RNAscope In Situ Hybridization for Apoe Gene

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RNAscope In Situ Hybridization was performed following manufacturer’s instructions. Mounted tissue sections were warmed on a 40°C hot plate for 15 minutes and then dehydrated with 50%, 70%, and 100% ethyl alcohol for 5 minutes at each gradient, followed by hydrogen peroxide (Cat No.322335 ACDBio) treatment for 10 minutes. After washing the tissue with Deionized (DI) water, tissue sections were placed in boiling Target Retrieval Reagent (Cat No.322380 ACDBio) for 15 minutes and then immediately transferred to DI water. Slides were then blocked in Protease III (Cat No.322337 ACDBio) for 30 minutes at 40 °C. Apoe probe (Cat No. 313271-C2) was then added for 2 hours at 40 °C within a humidity control chamber. Signal amplification and detection reagents (Cat No.322310 ACDBio) were applied sequentially and incubated in AMP 1, AMP 2, and AMP 3 for 30 minutes each. Before adding each AMP reagent, samples were washed twice with washing buffer (Cat NO.310091 ACDBio). Respective HRPs were placed on slides for 15 minutes at 40 °C followed by 30 minutes of respective Opal dye (FP1487001KT Akoya Biosciences) for 30 minutes at 40 °C and HRP blocker for 15 minutes at 40 °C.
Henningfield C.M., Arreola M.A., Soni N., Spangenberg E.E, & Green K.N. (2021). Microglia-specific ApoE knock-out does not alter Alzheimer’s Disease plaque pathogenesis or gene expression. Glia, 70(2), 287-302.
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2

Multiplex RNA-Protein Co-Detection in Brain

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We used the RNAscope Multiplex Fluorescent Reagent Kit v2 with the RNA-Protein Co-Detection Ancillary Kit for co-detection of mRNA and protein. For smFISH, we used custom-made RNAscope probes for Gad1, VGluT2 (Slc17a6), and NeuN (Rbfox3). Probes were based on the coding sequence of each gene, and single-nucleotide polymorphisms were included by alternating between species (P. maniculatus, P. polionotus). For IHC, we used the rabbit anti-c-Fos (1:100, Synaptic Systems, 226003) and rabbit anti-GFP (1:100, Thermo Fisher, A-11122) antibodies to detect c-Fos protein and the YFP tag in the viral vector, respectively, and HRP-labeled goat anti-rabbit antibody (1:500, PerkinElmer, NEF812001EA) for secondary detection. We visualized RNA probes and antibodies with Opal 520, Opal 570, and Opal 690 dyes (1:1000, Akoya Biosciences, FP1487001KT, FP1488001KT, FP1497001KT), and counterstained with DAPI. Regions of interest (mSC, dPAG) were imaged on a LSM 700 laser scanning confocal microscope (Zeiss), with Z-stacks of 21 slices spaced at 0.99 μm. We then used QuPath v0.2.3 to quantify the overlap of FISH and IHC signals in the maximum projection images.
Baier F., Reinhard K., Tong V., Murmann J., Farrow K, & Hoekstra H.E. (2023). The neural basis of defensive behaviour evolution in Peromyscus mice. bioRxiv.
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3

Fluorescent In-Situ Detection of pax6a and krtt1c19e

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We detected the expression of pax6a and krtt1c19e with the RNAScope Fluorescent V2 kit (323,110, BioTechne), using probes Dr-pax6a 532,481) and Dr-krtt1c19e (1117231-C2), and Opal dyes 520 (1:1000 dilution) and 620 (1:1500 dilution) (FP1487001KT, FP1495001KT, Akoya Biosciences). We performed the staining on 5-μm paraffin-embedded, formalin-fixed sections, using manufacturer’s protocol with an additional baking of sections at 60°C for 30 min after deparaffinization. We performed a 17-min target retrieval, followed by a 20-min protease treatment.
Ikkala K., Raatikainen S., Koivula H, & Michon F. (2022). Zebrafish cornea formation and homeostasis reveal a slow maturation process, similarly to terrestrial vertebrates’ corneas. Frontiers in Physiology, 13, 906155.
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4

Multiplex Immunohistochemistry Staining Protocol

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4 μm FFPE tissue sections were baked for 3 hrs. at 62 degrees Celsius in vertical slide orientation with subsequent deparaffinization performed on the Leica Bond RX followed by 30 minutes of antigen retrieval with Leica Bond ER2 followed by 6 sequential cycles of staining with each round including a 30-minute combined block and primary antibody incubation (PerkinElmer antibody diluent/block ARD1001).
Detection of all primary antibodies was performed using a goat anti-mouse Poly HRP secondary antibody or goat anti-rabbit Poly HRP secondary antibody (Invitrogen B40961/2; 10-minute incubation). The HRP-conjugated secondary antibody polymer was detected using fluorescent tyramide signal amplification using Opal dyes 520, 540, 570, 620, 650 and 690 (Akoya FP1487001KT, FP1494001KT, FP1488001KT, FP1495001KT, FP1496001KT, FP1497001KT). The covalent tyramide reaction was followed by heat induced stripping of the primary/secondary antibody complex using Perkin Elmer AR9 buffer (AR900250ML) and Leica Bond ER2 (90% ER2 and 10% AR9) at 100 degrees Celsius for 20 minutes preceding the next cycle. After 6 sequential rounds of staining, sections were stained with Hoechst (Invitrogen 33342) to visualize nuclei and mounted with ProLong Gold antifade reagent mounting medium (Invitrogen P36930).
Chan J.M., Quintanal-Villalonga Á., Gao V.R., Xie Y., Allaj V., Chaudhary O., Masilionis I., Egger J., Chow A., Walle T., Mattar M., Yarlagadda D.V., Wang J.L., Uddin F., Offin M., Ciampricotti M., Qeriqi B., Bahr A., de Stanchina E., Bhanot U.K., Lai W.V., Bott M.J., Jones D.R., Ruiz A., Baine M.K., Li Y., Rekhtman N., Poirier J.T., Nawy T., Sen T., Mazutis L., Hollmann T., Pe’er D, & Rudin C.M. (2021). Signatures of plasticity, metastasis, and immunosuppression in an atlas of human small cell lung cancer. Cancer cell, 39(11), 1479-1496.e18.
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5

RNAscope analysis of pituitary Ig expression

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Pituitary was isolated from perfused mouse and fixed with 4% Paraformaldehyde (PFA) (Solarbio, P1110) overnight at room temperature. Paraffin embedded tissues were sectioned at 5μm and processed to RNAscope. RNAscope was performed using the Multiplex Fluorescence v.2 kit (Advanced Cell Diagnostics, 323100) according to the manufacturer’s protocol. Probes for mouse Igkc (414291) and Igha (414281-C2) were commercially available from the manufacturer, and secondary Opal 520 reagents (FP1487001KT, Akoya Biosciences) were diluted 1:500 in TSA buffer.
Li Y., Wang J., Wang R., Chang Y, & Wang X. (2023). Gut bacteria induce IgA expression in pituitary hormone-secreting cells during aging. iScience, 26(10), 107747.
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6

Lung Development Analysis by FISH

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Lungs were isolated at E11.5 and immediately fixed in 4% paraformaldehyde in PBS for 24 hr at 4ºC. Lungs were then washed through a sucrose gradient, embedded in OCT (Tissue Tek), and frozen on dry ice. Sectioning was performed on a Leica CM3050S cryostat. Fluorescence in situ hybridization was performed using the standard RNAScope Multiplex Fluorescent V2 Assay (ACD) protocol for fixed-frozen samples. The probes were for Mus musculus Hoxb6 (RNAScope Probe 564171) and Ptn (RNAScope Probe 486381) and the fluorophores were Opal 520 and Opal 620 (Akoya Biosciences FP1487001KT and FP1495001KT). Sections were imaged on a Nikon A1RSi confocal microscope with a 20× objective.
Goodwin K., Jaslove J.M., Tao H., Zhu M., Hopyan S, & Nelson C.M. (2022). Patterning the embryonic pulmonary mesenchyme. iScience, 25(3), 103838.
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7

Multiplex Fluorescent In Situ Hybridization

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RNAscope probes were ordered from Advanced Cell Diagnostics (ACD) for the following genes: ERBB2, ACTG2, TAGLN, CTLA4, FOXP3, CD3D, CD4, CD8A, MA4A1, CD1C and CD68. Cryosections of two snap-frozen DCIS samples (OCT embedded) were cut at a thickness of 10um and used to performed RNA in situ hybridization assay with the RNAscope Multiplex Fluorescent v2 kit according to the manufacturer’s instructions (Cat# 323110 and 323120) with following modifications: tissue sections were fixed in 10% NBF for 1hr at 4°C, all washing steps were increased to 3 times (3–5min each wash), areas enclosed by hydrophobic barrier were 0.75”×0.75”, for all reagent steps 150ul were dispensed, tissues were treated with Protease III for 15min at RT, Opal dyes were used at 1/750–1/2250 (Akoya Biosciences FP1487001KT, FP1488001KT, FP1495001KT FP1497001KTP), kit DAPI stain was replaced by a 1/2000 working stock in PBS (Invitrogen D1306 in DMF, 5mg/ml), and slides were mounted in Prolong diamond (Invitrogen #P36970). Fluorescent Images were scanned using a motorized stage on the Nikon Eclipse Ti2 microscope with 20X objective and analyzed with the Nikon NIS-Elements AR software (5.30.04).
Wei R., He S., Bai S., Sei E., Hu M., Thompson A., Chen K., Krishnamurthy S, & Navin N.E. (2022). Spatial charting of single cell transcriptomes in tissues. Nature biotechnology, 40(8), 1190-1199.
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8

Multicolor RNA Expression Analysis in Mouse Striatum

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Sagittal brain slices (∼12 μm thick) of WT mice were used to detect Drd1, Adora2a, and Fxyd2 gene expression in the striatum. RNAscope Multiplex Fluorescent Reagent version 2 (No. 323100; ACD) was used with dyes and dilutions as follows: 1:1500 Drd1 (high expression gene)-Opal520, 1:1500 Adora2a (high expression gene)-Opal570 and 1:750 Fxyd2 (low expression gene)-Opal690 (Nos. FP1487001KT, FP1488001KT, and FP1497001KT, respectively; Akoya Bioscience), following HybEZ II Hybridization System (No. 321721) protocols.
Guerri L., Dobbs L.K., da Silva e Silva D.A., Meyers A., Ge A., Lecaj L., Djakuduel C., Islek D., Hipolito D., Martinez A.B., Shen P.H., Marietta C.A., Garamszegi S.P., Capobianco E., Jiang Z., Schwandt M., Mash D.C., Alvarez V.A, & Goldman D. (2022). Low Dopamine D2 Receptor Expression Drives Gene Networks Related to GABA, cAMP, Growth and Neuroinflammation in Striatal Indirect Pathway Neurons. Biological Psychiatry Global Open Science, 3(4), 1104-1115.
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9

Multiplexed RNA Detection in Mouse Brain

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RNAScope was performed on fresh frozen coronal brain sections (10μm thick) using the Multiplex Fluorescence v.2 kit (Advanced Cell Diagnostics) according to the manufacturer’s protocol with minor modifications. Tissue fixation with 4% PFA was extended to 60 min at RT, and Protease IV treatment was shortened to 20 min to better preserve the hippocampal tissue. Probes for mouse Pdgfrα, SRF, Fgfr3 and Fgf17 were commercially available from the manufacturer and secondary Opal 690 and 520 reagents (FP1497001KT and FP1487001KT, Akoya Biosciences) were diluted at 1:1500 in TSA buffer.
Iram T., Kern F., Kaur A., Myneni S., Morningstar A.R., Shin H., Garcia M.A., Yerra L., Palovics R., Yang A.C., Hahn O., Lu N., Shuken S.R., Haney M.S., Lehallier B., Iyer M., Luo J., Zetterberg H., Keller A., Zuchero J.B, & Wyss-Coray T. (2022). Young CSF restores oligodendrogenesis and memory in aged mice via Fgf17. Nature, 605(7910), 509-515.
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10

Visualization of Wnt6 mRNA in Corneal Tissue

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The RNAscope V2 Fluorescent Assay (ACD Biosystems, Newark, CA) was performed according to the ACD Biosystems protocol for fresh-frozen tissue. Corneal sections from 3 donors were hybridized with the Wnt6 mRNA probe. Assays were performed in 2 independent experiments for each donor. To confirm the mRNA integrity of the tissues, positive control probes targeting human housekeeping genes Polr2a, PPIB, and HPRT were visualized (Supplemental Fig. 1). The probes were amplified according to the manufacturer’s instructions and labeled with the fluorophore Opal 520 nm (Akoya Biosciences, FP1487001KT, 1:250), Opal 570 nm (Akoya Biosciences, FP1488001KT, 1:1500), and Opal 690 nm (Akoya Biosciences, SKU FP1497001KT, 1:1500). The Olympus FluoView FV1000 was used to visualize the fluorescence in situ hybridization signals. Quantitation of the dots among the limbal layers was performed with Imaris software and the surface and dots functions (V. 9.7.0, Imaris, Oxford Instruments, Oxon, UK).
Bonnet C., Oh D., Mei H., Robertson S., Chang D., Bourges J.L., Behar-Cohen F., Zheng J.J, & Deng S.X. (2021). Wnt6 plays a complex role in maintaining human limbal stem/progenitor cells. Scientific Reports, 11, 20948.
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