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Epitope retrieval solution

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Epitope retrieval solution is a laboratory reagent used to expose or unmask antigenic sites (epitopes) on tissue samples. It is a critical step in immunohistochemistry (IHC) procedures, allowing the antibodies to bind to their target proteins more effectively for visualization and analysis.

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Lab products found in correlation

Alexa fluor 488 or 555, by Thermo Fisher Scientific (2 mentions) Anti phospho histone h3 ser10, by Merck Group (2 mentions) Sc 47739, by Merck Group (2 mentions) Anti phospho histone gamma h2ax, by Merck Group (2 mentions) Ab62623, by Abcam (2 mentions) Antifade mounting medium, by Vector Laboratories (2 mentions) Ab68167, by Abcam (2 mentions) Anti aurora b, by Merck Group (2 mentions) Mayer s hematoxylin, by Merck Group (1 mentions) Aquamount, by Thermo Fisher Scientific (1 mentions) Bx43 microscope, by Olympus (1 mentions) 3 3 diaminobenzidine (dab), by Merck Group (1 mentions) Cellsens software, by Olympus (1 mentions) Vimentin, by Santa Cruz Biotechnology (1 mentions) Tgf β, by Santa Cruz Biotechnology (1 mentions) Betazoid dab chromogen kit, by Biocare Medical (1 mentions) Formalin solution, by Merck Group (1 mentions) Fluoview fv1000 confocal microscope, by Olympus (1 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Metamorph software, by Molecular Devices (1 mentions)

Spelling variants of this product

IHC-Tek epitope retrieval solution (14 citations) Epitope retrieval solution and steamer (7 citations) IHC-TekTM Epitope Retrieval Solution (2 citations)

9 protocols using epitope retrieval solution

1

Immunohistochemical Analysis of BMPR2 and BMP4 in PAH

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Paraffin sections of the lung and heart tissues of the PAH mice and control mice were baked for 40 min, and placed into the reagent tank for dewaxing, hydration, and incubation with 3% of hydrogen peroxide (diluted with pure water) for 10 minutes to eliminate the activity of endogenous peroxidase. Paraffin sections were then deparaffinized and rehydrated. Antigen retrieval was performed using heat-incubation in epitope retrieval solution (IHC World, USA) for 30 minutes. The primary antibodies used for immunohistochemistry were anti-BMPR2 (1:200) and anti-BMP4 (1:50) antibodies from Cell Signaling Technology, USA.
Li N., Zhu L., Zhu C., Zhou H., Zheng D., Xu G., Shi H., Gao J., Li A.J., Wang Z., Sun L., Li X, & Shao G. (2021). BMPR2 promoter methylation and its expression in valvular heart disease complicated with pulmonary artery hypertension. Aging (Albany NY), 13(22), 24580-24604.
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2

Immunohistochemistry of Cardiac Tissues

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Tissues were fixed in 4% paraformaldehyde in PBS overnight at 4°C
and incubated in 30% sucrose in PBS at 4°C overnight. Tissues were
embedded in tissue freezing medium and cut 8 μm thicknesses. For antigen
retrieval, either 1 mM EDTA with 0.05% Tween 20 in boiling water or epitope
retrieval solution (IHC World) in a steamer (IHC-Tek Epitope Retrieval Streamer
Set) were used, then sections were blocked with 10% serum from the host animal
of secondary antibodies, and incubated with primary anti-bodies overnight at 4
°C. The sections were subsequently washed with PBS and incubated with
corresponding secondary antibodies conjugated to Alexa Fluor 488 or 555
(Invitrogen). The slides were mounted in antifade mounting medium (Vector
Laboratories, Burlingame, California). Primary antibodies used are as follows:
anti-phospho histone H3 Ser10 (Millipore 06–570, 1:100), anti-aurora B
(Sigma A5102, 1:100), anti-troponin T, cardiac isoform Ab-1, clone 13–11
(Thermo Scientific MS-295-P1, 1:100), anti-sarcomeric α-actinin (Abcam,
ab68167, 1:100), anti-8 hydroxyguanosine (Abcam ab62623, 1:50),
anti-phosphorylated ATM (Santa Cruz Biotechnology sc-47739, 1:100),
Anti-phospho-Histone gamma H2AX (Millipore-Sigma 05–636). DAPI was used
for the nuclear staining.
Cardoso A.C., Lam N.T., Savla J.J., Nakada Y., Pereira A.H., Elnwasany A., Menendez-Montes I., Ensley E.L., Petric U.B., Sharma G., Sherry A.D., Malloy C.R., Khemtong C., Kinter M.T., Tan W.L., Anene-Nzelu C.G., Foo R.S., Nguyen N.U., Li S., Ahmed M.S., Elhelaly W.M., Abdisalaam S., Asaithamby A., Xing C., Kanchwala M., Vale G., Eckert K.M., Mitsche M.A., McDonald J.G., Hill J.A., Huang L., Shaul P.W., Szweda L.I, & Sadek H.A. (2020). Mitochondrial Substrate Utilization Regulates Cardiomyocyte Cell Cycle Progression. Nature metabolism, 2(2), 167-178.
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3

Immunohistochemical Analysis of Small Intestine

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Formalin-fixed, paraffin-embedded small intestine tissue sections were deparaffinized using standard laboratory protocol. Epitope retrieval solution and steamer (IHC-World, Woodstock, MD, USA) were used for antigen epitope retrieval of the tissue sections. 3% H2O2 solution was used to block the endogenous peroxidase activity for 5 minutes, followed by serum blocking (5% goat serum, 1hr). The primary antibody for α-SMA (Abcam, Cambridge, MA), TGF-β, vimentin (Santa Cruz Biotechnology, Dallas, TX) were used overnight in recommended dilutions (1:300). Species-specific biotinylated conjugated secondary antibody and streptavidin-conjugated with HRP were used to perform antigen-specific immunohistochemistry according to the manufacturer’s standard protocols. 3, 3’ Diaminobenzidine (DAB) (Sigma-Aldrich, St. Louis, and MA) was used as a chromogenic substrate. Tissue sections were counter-stained with Mayer’s hematoxylin (Sigma- Aldrich). Tissue sections were washed with 1X PBS-T (Phosphate buffered saline+ 0.05% Tween 20) between the steps. Sections were finally mounted in Aqua mount (Fisher Scientific) and observed under a 20X objective using an Olympus BX43 microscope (Olympus, America). Morphometric analysis was done using CellSens Software from Olympus America (Center Valley, PA).
Sarkar S., Saha P., Seth R.K., Mondal A., Bose D., Kimono D., Albadrani M., Mukherjee A., Porter D.E., Scott G.I., Xiao S., Brooks B., Ferry J., Nagarkatti M., Nagarkatti P, & Chatterjee S. (2020). Higher intestinal and circulatory lactate associated NOX2 activation leads to an ectopic fibrotic pathology following microcystin co-exposure in murine fatty liver disease. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 238, 108854.
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4

Immunohistochemical Analysis of Bcl-2

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Sections were baked for 40 min, and placed into the reagent tank for dewaxing, hydration, and incubation with 3% hydrogen peroxide (diluted with pure water) for 10 min to eliminate the activity of endogenous peroxidase. Paraffin-sections were then deparaffinized and rehydrated. Antigen retrieval was performed using heat-incubation in epitope retrieval solution (IHC World, U.S.A.) for 30 min. The primary antibodies used for immunohistochemistry were anti-bcl-2 (1:200).
Li N., Zhu L., Zhou H., Zheng D., Xu G., Sun L., Gao J, & Shao G. (2020). miRNA-1183-targeted regulation of Bcl-2 contributes to the pathogenesis of rheumatic heart disease. Bioscience Reports, 40(11), BSR20201573.
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5

Immunohistochemistry of Cardiac Tissues

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Tissues were fixed in 4% paraformaldehyde in PBS overnight at 4°C
and incubated in 30% sucrose in PBS at 4°C overnight. Tissues were
embedded in tissue freezing medium and cut 8 μm thicknesses. For antigen
retrieval, either 1 mM EDTA with 0.05% Tween 20 in boiling water or epitope
retrieval solution (IHC World) in a steamer (IHC-Tek Epitope Retrieval Streamer
Set) were used, then sections were blocked with 10% serum from the host animal
of secondary antibodies, and incubated with primary anti-bodies overnight at 4
°C. The sections were subsequently washed with PBS and incubated with
corresponding secondary antibodies conjugated to Alexa Fluor 488 or 555
(Invitrogen). The slides were mounted in antifade mounting medium (Vector
Laboratories, Burlingame, California). Primary antibodies used are as follows:
anti-phospho histone H3 Ser10 (Millipore 06–570, 1:100), anti-aurora B
(Sigma A5102, 1:100), anti-troponin T, cardiac isoform Ab-1, clone 13–11
(Thermo Scientific MS-295-P1, 1:100), anti-sarcomeric α-actinin (Abcam,
ab68167, 1:100), anti-8 hydroxyguanosine (Abcam ab62623, 1:50),
anti-phosphorylated ATM (Santa Cruz Biotechnology sc-47739, 1:100),
Anti-phospho-Histone gamma H2AX (Millipore-Sigma 05–636). DAPI was used
for the nuclear staining.
Cardoso A.C., Lam N.T., Savla J.J., Nakada Y., Pereira A.H., Elnwasany A., Menendez-Montes I., Ensley E.L., Petric U.B., Sharma G., Sherry A.D., Malloy C.R., Khemtong C., Kinter M.T., Tan W.L., Anene-Nzelu C.G., Foo R.S., Nguyen N.U., Li S., Ahmed M.S., Elhelaly W.M., Abdisalaam S., Asaithamby A., Xing C., Kanchwala M., Vale G., Eckert K.M., Mitsche M.A., McDonald J.G., Hill J.A., Huang L., Shaul P.W., Szweda L.I, & Sadek H.A. (2020). Mitochondrial Substrate Utilization Regulates Cardiomyocyte Cell Cycle Progression. Nature metabolism, 2(2), 167-178.
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6

Tissue Harvesting and Immunohistochemistry Protocol

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Tissue harvest and fixation: At the end of the study, rats were anesthetized with isoflurane (2-5%) and tissues excised and placed in 10% formalin solution (Sigma; St Louis, MO) for 48 hours before being paraffin embedded, blocked and processed (Augusta University Core facility). Kidneys were paraffin embedded in an automatic tissue processor and 3-μm cut sections mounted on siliconized/charged slides. The slides were deparaffinized and hydrated and antigen retrieval performed using IHC-Tek Epitope retrieval solution at steaming for 40 min (IHCWorld, cat#IW-1100). Tissue was blocked with 3% hydrogen peroxide in methanol for 10 min. The primary antibody used are listed in the Table 3. The goat anti-rabbit IgG-HRP conjugated secondary antibody (Santa-cruz Cat#sc-2004, 400ug/ml, 1:400 at 1ug/ml) was used for 30 min at room temperature. The slides were stained with Betazoid DAB Chromogen Kit (Biocare Medical Cat#BDB2004H). Omitting the first antibody served as a negative control and resulted in no positive staining in tissue. All end-point analysis was blinded to the investigators. For histological scoring all identifiers were removed and slides given a number before being scored by an investigator that was unaware of the hypothesis being tested. Data were then compiled by the primary investigator who had access to the numbering key.
Ray S.C., Baban B., Tucker M.A., Seaton A.J., Chang K.C., Mannon E.C., Sun J., Patel B., Wilson K., Musall J.B., Ocasio H., Irsik D., Filosa J.A., Sullivan J.C., Marshall B., Harris R.A, & O'Connor P.M. (2018). Oral NaHCO3 activates a splenic anti-inflammatory pathway; evidence cholinergic signals are transmitted via mesothelial cells. Journal of immunology (Baltimore, Md. : 1950), 200(10), 3568-3586.
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7

Immunofluorescent Detection of TIMM13 and Amyloid-β

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Paraffin-sections were rehydrated and boiled in epitope retrieval solution (IHC World, Woodstock, MD, USA), followed by incubation with antibodies against TIMM13 (Novus, USA; NBP2-13431). Antigen-antibody complexes were visualized using the Alexa Fluor conjugated secondary antibody (Invitrogen). Aβ plaques were stained with 0.01 mg/mL Methoxy-X04 for 30 min. Fluorescence images were obtained using a Fluoview FV 1000 confocal microscope (Olympus, Japan) with FV10-MSASW software and analyzed using MetaMorph software (Molecular Devices, San Jose, CA, USA).
Kim S.H., Choi K.Y., Park Y., McLean C., Park J., Lee J.H., Lee K.H., Kim B.C., Huh Y.H., Lee K.H, & Song W.K. (2021). Enhanced Expression of microRNA-1273g-3p Contributes to Alzheimer’s Disease Pathogenesis by Regulating the Expression of Mitochondrial Genes. Cells, 10(10), 2697.
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8

Immunometabolism Profiling in AAV Kidney

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The expression and localisation of immunometabolism-associated proteins was evaluated in paraffin-embedded kidney sections by indirect immunofluorescence in five patients with AAV. Kidney biopsies were classified based on histology as focal, crescentic, mixed or sclerotic, according to consensus guidelines.32 (link) Briefly, after xylene/ethanol deparaffinisation, tissue sections were pretreated with Epitope Retrieval Solution (IHC World, Woodstock, Maryland, USA) for 40 min at 60°C. Subsequently, non-specific binding was blocked with 10% Normal Goat Serum (NGS) for 30 min at room temperature. Tissue slides were double stained with combinations of antibodies specific for CD68, TKT and HIF-1α (Abcam, Cambridge, Massachusetts, USA; BioLegend, San Diego, California, USA) overnight at 4°C in 1% NGS. Secondary antibodies were purchased from ThermoFisher Scientific (Grand Island, New York, USA), and tissue sections incubated accordingly for 1 hour at room temperature in 1% NGS. Hoechst-stained slides were then mounted with ProLong Gold Antifade Mountant (ThermoFisher), and visualised after curing overnight.
Grayson P.C., Eddy S., Taroni J.N., Lightfoot Y.L., Mariani L., Parikh H., Lindenmeyer M.T., Ju W., Greene C.S., Godfrey B., Cohen C.D., Krischer J., Kretzler M, & Merkel P.A. (2018). Metabolic pathways and immunometabolism in rare kidney diseases. Annals of the rheumatic diseases, 77(8), 1226-1233.
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9

Histological Analysis of Tissue Samples

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Specimens were fixed in 4% paraformaldehyde (PFA), dehydrated and paraffin-embedded. Histological sections of 5 μm thickness were obtained using a microtome. Hematoxylin-eosin (H&E) staining was performed using a standard protocol. Stained slices were visualized using conventional microscopy and recorded using Axio Vision 4 image processing software (Carl Zeiss Microscopy, White Plains, NY, USA). Nuclear staining was performed with DAPI (Thermo Fisher Scientific, Waltham, MA, USA). CD31 staining was performed using a monoclonal rabbit anti-rat antibody at a dilution of 1:100 (Abcam, Cambridge, UK) in combination with a secondary polyclonal Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Abcam, Cambridge, UK). For antigen retrieval, sections were boiled in Epitope Retrieval Solution (IHC World, Woodstock, GA, USA).
Falkner F., Mayer S.A., Thomas B., Zimmermann S.O., Walter S., Heimel P., Thiele W., Sleeman J.P., Bigdeli A.K., Kiss H., Podesser B.K., Kneser U., Bergmeister H, & Schneider K.H. (2023). Acellular Human Placenta Small-Diameter Vessels as a Favorable Source of Super-Microsurgical Vascular Replacements: A Proof of Concept. Bioengineering, 10(3), 337.
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