Histostain sp kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Histostain-SP kit is a reagent set used for the detection of target antigens in tissue sections. It provides a sensitive and specific immunohistochemical staining method for the visualization of antigens of interest in paraffin-embedded or frozen tissue samples.

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Lab products found in correlation

Alexa fluor 488 594 and cy5 coupled secondary antibodies, by Thermo Fisher Scientific (3 mentions) Non immune goat serum, by Thermo Fisher Scientific (3 mentions) Bx53 microscope, by Olympus (3 mentions) Alexa fluor 488 and 568 coupled secondary antibodies, by Thermo Fisher Scientific (2 mentions) Fluorescence microscope, by Olympus (2 mentions) Biotinylated universal secondary antibody, by Thermo Fisher Scientific (2 mentions) Nanozoomer 2.0 ht system, by Hamamatsu Photonics (2 mentions) Triton x 100, by Merck Group (2 mentions) Clearmount, by Thermo Fisher Scientific (2 mentions) Dab substrate, by Agilent Technologies (2 mentions) Pt link system, by Agilent Technologies (2 mentions) Nanozoomer 2.0 rs slide scanner, by Hamamatsu Photonics (1 mentions) Anti synapsin 1, by Cell Signaling Technology (1 mentions) Anti psd95, by Cell Signaling Technology (1 mentions) Anti trkb antibody, by Abcam (1 mentions) Anti synaptotagmin, by Merck Group (1 mentions) Igg2a, by Abcam (1 mentions) Hoechst, by Abcam (1 mentions) Fluorescent microscope, by Olympus (1 mentions) Aβ1 42, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Histostain kit (13 citations) Histostain SP Broad Spectrum kit (11 citations) SP Kit (5 citations) Histostain-SP (2 citations)

47 protocols using histostain sp kit

Immunohistochemistry and Immunofluorescence Staining

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Immunohistochemistry was performed by using the peroxidase protocol. Briefly, tissue sections were deparaffinized in xylene, hydrated through descending ethanol concentrations, and endogenous peroxidase activity was eliminated by incubation in 3% hydrogen peroxide in methanol for 5 min. After antigen-retrieval in boiling sodium citrate buffer (10 mM), the sections were incubated with primary antibodies for overnight at 4 °C. The signal was developed using Histostain-SP kit (Invitrogen). For the double immunofluorescence staining, the sections were incubated overnight at 4 °C with a mixture of antibodies. After being washed with TBS, the sections were incubated with a mixture of Alexa Fluor 488-/594- and CY5-coupled secondary antibodies (Invitrogen) for detection. Images were acquired through Confocal (Olympus FV1000).

Wang Z.H., Xia Y., Liu P., Liu X., Edgington-Mitchell L., Lei K., Yu S.P., Wang X.C, & Ye K. (2021). ApoE4 Activates C/EBPβ/δ-secretase with 27-hydroxycholesterol, Driving the Pathogenesis of Alzheimer’s Disease. Progress in neurobiology, 202, 102032.

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Immunohistochemical Staining of Teratomas and Arteries

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Teratomas and human arteries were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Sections were cut and mounted on positively charged slides. Sections were deparaffinized with xylene and rehydrated in a graded ethanol series. Endogenous peroxidase was quenched by placing the slides in 3% hydrogen peroxide in methanol for 10 min. Heat-induced epitope retrieval was performed by boiling the slides for 6 min in citrate buffer solution (pH 6.0) (Invitrogen). Slides were washed with phosphate-buffered saline and then stained with the monoclonal antibody MAB2909, which recognizes alkaline phosphatase (R&D Systems), using the Histostain-SP Kit (Invitrogen) according to the manufacturer’s instructions. In brief, after exposure to serum blocking solution, the sections were incubated successively with primary antibody, biotinylated secondary antibody, and streptavidin–horseradish peroxidase (HRP) conjugate. Visualization was performed with 3,3′-diaminobenzidine (Molecular Probes), which generates a brown-colored oxidation product upon reaction with the HRP-labeled streptavidin. Nuclei were stained with hematoxylin. For images, slides were scanned by NanoZoomer 2.0-RS slide scanner (Hamamatsu Photonics).

Jin H., Hilaire C.S., Huang Y., Yang D., Dmitrieva N.I., Negro A., Schwartzbeck R., Liu Y., Yu Z., Walts A., Davaine J.M., Lee D.Y., Donahue D., Hsu K.S., Chen J., Cheng T., Gahl W., Chen G, & Boehm M. (2016). Increased activity of TNAP compensates for reduced adenosine production and promotes ectopic calcification in the genetic disease ACDC. Science signaling, 9(458), ra121.

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Alzheimer's Disease Model in 5xFAD Mice

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5xFAD mice were obtained from Jackson lab and were bred in accordance with Emory Medical
School guidelines. The 5xFAD mice received vehicle or CF₃CN dissolved in 5%
DMSO/0.5% methylcellulose at a dose of 3 or 10 mg/kg/day. Mice were housed in AAALAC
(American Association for Accreditation of Laboratory Animal Care)-Accredited facilities,
and this study was approved by the institutional Animal Care and Use Committees at Emory
University.
Anti-TrkB antibody was bought from Biovision (Milpitas, CA). Antiphospho-TrkB Y816
antibody was raised against [H]-CKLQNLAKASPV-pY-LDILG-[OH] (a.a. 806–822) (EM437
and EM438) as rabbit polyclonal antibody. Anti-synaptotagmin, anti-Aβ, and
antitubulin were from Sigma-Aldrich (St Louis, MO). Anti-Akt, anti-p-Akt, anti-ERK,
anti-p-ERK1/2, anti-synapsin I, anti-PSD95, and antispinophilin antibodies were bought
from Cell Signaling (Boston, MA). The Histostain-SP kit and the Aβ 1-42 ELISA kit
was from Invitrogen (Grand Island, NY). All chemicals not included above were purchased
from Sigma-Aldrich.

Chen C., Ahn E.H., Liu X., Wang Z.H., Luo S., Liao J, & Ye K. (2021). Optimized TrkB Agonist Ameliorates Alzheimer’s Disease Pathologies and Improves Cognitive Functions via Inhibiting Delta-Secretase. ACS Chemical Neuroscience, 12(13), 2448-2461.

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Quantification of TIMP2 and MMP2 Expression

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Briefly, first, the samples were decalcified in 10% EDTA, and then embedded in paraffin. After the slices were created, the Histostain-SP Kit (Invitrogen) was used to evaluate the effects according to the manufacturer’s instructions. The primary antibodies against TIMP2 (Abcam, IgG2a, Mouse monoclonal, 1:250) and MMP2 (Abcam, IgG, Rabbit monoclonal, 1:250) were applied to slides, followed by goat anti-mouse IgG Alexa Fluor 596 (Abcam, IgG, Goat polyclonal, 1:400) or goat anti-rabbit IgG Alexa Fluor 647(Abcam, IgG, Goat polyclonal, 1:400). Finally, the sections were stained with Hoechst (11,000, Abcam) prior to be imaged by fluorescent microscope (Olympus). Then, the positive cell ratios of TIMP2 and MMP2 to evaluate expression levels were obtained by dividing the number of TIMP2- and MMP2-positive cells by the total number of cells in the defective area, respectively. The quantitative results were analyzed using ImageJ software with 3 randomly selected fields of each section and 3 sections per specimen.

Chen M., Zhou M., Fu Y., Li J, & Wang Z. (2021). Effects of miR-672 on the angiogenesis of adipose-derived mesenchymal stem cells during bone regeneration. Stem Cell Research & Therapy, 12, 85.

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Immunohistochemical Analysis of Amphiphysin I

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Free-floating 30-μm-thick serial sections were treated with 0.3% H₂O₂ for 10 min and washed three times in PBS. Then, sections were blocked in 1% BSA, 0.3% Triton X-100, for 30 min and incubated with anti-Amphiphysin I N278 (generated and verified as described in the main text, 1:1000), AT8, or AT100 overnight at 4 ℃. The signal was developed using a Histostain-SP kit (Invitrogen). The images were analyzed by Image J, plus the IHC Profiler plugin. The program counts the pixels and evaluates the percentage contribution of high positive, positive, low positive, and negative. The ‘optical density score’ was calculated as (percentage contribution of high positive*4 + percentage contribution of positive*3 + percentage contribution of low positive*2 + percentage contribution of negative*1)/100.

Zhang X., Zou L., Meng L., Xiong M., Pan L., Chen G., Zheng Y., Xiong J., Wang Z., Duong D.M., Zhang Z., Cao X., Wang T., Tang L., Ye K, & Zhang Z. (2021). Amphiphysin I cleavage by asparagine endopeptidase leads to tau hyperphosphorylation and synaptic dysfunction. eLife, 10, e65301.

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Immunohistochemical Evaluation of Bone Markers

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Immunohistochemistry was performed according to a previously reported study38 (link). Briefly, the samples were first decalcified in 10% ethylene diamine tetraacetic acid (EDTA) and then embedded in paraffin. After the sections were created, a Histostain-SP Kit (Invitrogen) was used according to the manufacturer’s instruction, primary antibodies against SMAD4, Osterix and Runx2 (all from Abcam) were applied to the slides. Positive cell ratios of SMAD4, Runx2 and Osterix were calculated to evaluate the expression levels by dividing SMAD4-, Runx2- and Osterix-positive cell number by total cell number within defect region, respectively.

Xie Q., Wei W., Ruan J., Ding Y., Zhuang A., Bi X., Sun H., Gu P., Wang Z, & Fan X. (2017). Effects of miR-146a on the osteogenesis of adipose-derived mesenchymal stem cells and bone regeneration. Scientific Reports, 7, 42840.

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Immunohistochemistry and Immunofluorescence Staining

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Immunohistochemistry was performed by using the peroxidase protocol. Briefly, tissue sections were deparaffinized in xylene, hydrated through descending ethanol concentrations, and endogenous peroxidase activity was eliminated by incubation in 3% hydrogen peroxide in methanol for 5 min. After antigen-retrieval in boiling sodium citrate buffer (10 mM), the sections were incubated with primary antibodies for overnight at 4 °C. The signal was developed using Histostain-SP kit (Invitrogen). For the double immunofluorescence staining, the sections were incubated overnight at 4 °C with a mixture of antibodies. After being washed with TBS, the sections were incubated with a mixture of Alexa Fluor 488-/594-and CY5-coupled secondary antibodies (Invitrogen) for detection. Images were acquired through Confocal (Olympus FV1000).

Wang Z.H., Xiang J., Liu X., Yu S.P., Manfredsson F.P., Sandoval I.M., Wu S., Wang J.Z, & Ye K. (2019). Deficiency in BDNF/TrkB Neurotrophic Activity Stimulates δ-Secretase by Upregulating C/EBPβ in Alzheimer’s Disease. Cell reports, 28(3), 655-669.e5.

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Antibody sources and characterization

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δ-Secretase antibody (clone 6E3 and 11B7) was a present from C. Watts, University of Dundee. TrkB antibody was from BioVision (Milpitas, CA). Phospho-TrkB Y816 antibody was raised against [H]-CKLQNLAKASPV-pY-LDILG-[OH] (amino acids 806 to 822) (EM437 and EM438) as rabbit polyclonal antibody. 4-HNE antibody was from Abcam (San Francisco, CA). Aβ, AT8, AT100, Tau 5, tubulin, and β-actin antibody were from Sigma-Aldrich (St. Louis, MO). C/EBPβ (H-7) antibody was bought from Santa Cruz Biotechnology (Dallas, TX). Legumain (D6S4H) and p-C/EBPβ antibodies were bought from Cell Signaling Technology (Boston, MA). Histostain-SP kit, Aβ1-42, and Aβ1-40 ELISA kit were from Invitrogen (Grand Island, NY). TNF-α, IL-1β, and IL-6 ELISAs were from eBioscience (San Diego, CA). All chemicals not included above were purchased from Sigma-Aldrich.

Chen C., Ahn E.H., Kang S.S., Liu X., Alam A, & Ye K. (2020). Gut dysbiosis contributes to amyloid pathology, associated with C/EBPβ/AEP signaling activation in Alzheimer’s disease mouse model. Science Advances, 6(31), eaba0466.

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Visualizing Amyloid-Beta and Microglia

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Free-floating 30-μm-thick serial sections were treated with 0.3% hydrogen peroxide for 10 min, and then incubated with anti-Aβ antibody or anti-IBA1 antibody (1:500) overnight. The signal was developed using Histostain-SP kit (#956543, invitrogen) according to the manufacturer's instructions.

Zhang Z., Obianyo O., Dall E., Du Y., Fu H., Liu X., Kang S.S., Song M., Yu S.P., Cabrele C., Schubert M., Li X., Wang J.Z., Brandstetter H, & Ye K. (2017). Inhibition of delta-secretase improves cognitive functions in mouse models of Alzheimer's disease. Nature Communications, 8, 14740.

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Immunohistochemistry and Immunofluorescence Staining

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Immunohistochemistry was performed by using the peroxidase protocol. Briefly, tissue sections were deparaffinized in xylene, hydrated through descending ethanol, and endogenous peroxidase activity was eliminated by incubation in 3% hydrogen peroxide in methanol for 5 min. After antigen-retrieval in boiling sodium citrate buffer (10 mM), the sections were incubated with primary antibodies for overnight at 4 °C. The signal was developed using Histostain-SP kit (Invitrogen). For the double immunofluorescence staining, the sections were incubated overnight at 4 °C with a mixture of antibodies. After being washed with TBS, the sections were incubated with a mixture of Alexa Fluor 488- and 568-coupled secondary antibodies (Invitrogen) for detection. Images were acquired through Confocal (Olympus FV1000).

Wang Z.H., Liu P., Liu X., Manfredsson F.P., Sandoval I.M., Ping Yu S., Wang J.Z, & Ye K. (2017). Delta-secretase Phosphorylation by SRPK2 Enhances its Enzymatic Activity, Provoking the Pathogenesis in Alzheimer’s Disease. Molecular cell, 67(5), 812-825.e5.

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