Citric acid based antigen unmasking solution

Manufactured by Vector Laboratories

Sourced in Canada, United States

Citric acid-based antigen unmasking solution is a laboratory reagent designed to facilitate the detection of target antigens in biological samples. It functions by exposing epitopes that may be obscured during sample preparation, allowing for improved immunodetection of the desired analytes.

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Lab products found in correlation

Vectastain elite abc reagent, by Vector Laboratories (3 mentions) Hematoxylin, by Merck Group (3 mentions) Eclipse ti u microscope, by Nikon (3 mentions) Immpact dab peroxidase substrate system, by Vector Laboratories (3 mentions) Biotinylated donkey anti mouse secondary antibody, by Jackson ImmunoResearch (2 mentions) Anti kitlg sc 13126, by Santa Cruz Biotechnology (2 mentions) Mouse on mouse blocking reagent, by Vector Laboratories (2 mentions) Superfrost plus glass slide, by Thermo Fisher Scientific (1 mentions) 3 3 diaminobenzidine dab peroxidase substrate kit, by Vector Laboratories (1 mentions) Goat anti rabbit igg secondary antibody, by Vector Laboratories (1 mentions) Dp72 light microscope, by Olympus (1 mentions) Ab125212, by Abcam (1 mentions) Ab32536, by Abcam (1 mentions) At2 slide scanner, by Leica (1 mentions) Biotinylated secondary antibody, by Vector Laboratories (1 mentions) Dpx mountant, by Merck Group (1 mentions) Vectastain elite abc hrp kit, by Merck Group (1 mentions) Sigma fast dab tablets, by Merck Group (1 mentions) Antibody diluent, by Zytomed Systems (1 mentions) Axio scan z1 slide scanner, by Zeiss (1 mentions)

Spelling variants of this product

Antigen unmasking solution (267 citations) Unmasking solution (21 citations) Citric acid-based unmasking solution (2 citations) Citric acid (2 citations)

23 protocols using citric acid based antigen unmasking solution

Immunohistochemical Analysis of PLZF and EGR1

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Briefly, paraformaldehyde-fixed tissues were processed and embedded in paraffin as previously described [62 (link)]. Tissue blocks were serially sectioned into 5-μm thick sections that were placed on Superfrost Plus glass slides (Fisher Scientific, Inc., Pittsburgh, PA). For immunohistochemical analyses, sections were deparaffinized before being rehydrated and boiled in a citric acid-based antigen unmasking solution (Vector Laboratories, Inc., Burlingame, CA). After blocking, sections were incubated overnight at 4°C with a rabbit polyclonal anti-human PLZF (H-300) antibody (1:150 dilution; sc-22839, Santa Cruz Biotechnology Inc., Dallas, TX) or anti-human EGR1 (#15F7) antibody (1:150 dilution; Cell Signaling, Danvers, MA). After incubation with the primary antibodies, sections were incubated with the goat anti-rabbit IgG secondary antibody (Vector Laboratories, Inc.), followed by incubation with ZyMax streptavidin-horseradish peroxidase conjugate (Invitrogen Corporation, Carlsbad, CA). The 3, 3’-diaminobenzidine (DAB) peroxidase substrate kit (Vector Laboratories, Inc.) was used to visualize immunoreactivity before sections were counterstained with hematoxylin. Cover slips were mounted onto stained sections using Slowfade mounting media (Fisher Scientific, Inc.).

Kommagani R., Szwarc M.M., Vasquez Y.M., Peavey M.C., Mazur E.C., Gibbons W.E., Lanz R.B., DeMayo F.J, & Lydon J.P. (2016). The Promyelocytic Leukemia Zinc Finger Transcription Factor Is Critical for Human Endometrial Stromal Cell Decidualization. PLoS Genetics, 12(4), e1005937.

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Immunohistochemical Analysis of Maxilla Sections

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The slides containing the maxilla sections were submerged in a citric acid-based antigen unmasking solution (Vector Laboratories) at 65°C overnight for antigen retrieval and then incubated with anti-CD68 antibody (cat. no. ab125212; Abcam) or p65 (cat. no. ab32536; Abcam) as primary antibodies. The tissue sections were visualized using 3-amino-9-ethylcarbazole (AEC; cat. no. SK-4205; Vector Laboratories, Inc.) at room temperature for 1 min, followed by counterstaining with hematoxylin for 1 min at room temperature, then sealed with ImmunoHistoMount^™ solution (Agilent Technologies, Inc.). The digital images of the stained sections were obtained using the DP72 light microscope (Olympus Corporation).

Suh J.S., Kim S.Y., Lee S.H., Kim R.H, & Park N.H. (2022). Hyperlipidemia is necessary for the initiation and progression of atherosclerosis by severe periodontitis in mice. Molecular Medicine Reports, 26(2), 273.

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TNBC Xenograft Tumor Tissue Analysis

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TNBC xenograft tumor tissues were fixed in neutral-buffered formalin and
embedded in paraffin. Sections (5 μm thick) were deparaffinized in
xylene, rehydrated in graded alcohols, and washed in distilled water. Antigens
were retrieved by boiling the sections in citric acid–based antigen
unmasking solution (Vector, Burlingame, CA) for 30 min. Immunohistochemistry
(IHC) staining was performed using the Lab Vision automated system (Thermo
Fisher) through the Division of Surgery Histology Core. Immunostained slides
were scanned by using an Aperio AT2 slide scanner, and images were captured
using the Aperio Image Scope V12 (Leica, Buffalo Grove, IL).

Lee J., Lim B., Pearson T., Choi K., Fuson J.A., Bartholomeusz C., Paradiso L.J., Myers T., Tripathy D, & Ueno N.T. (2019). Anti-tumor and anti-metastasis efficacy of E6201, a MEK1 inhibitor, in preclinical models of triple-negative breast cancer. Breast cancer research and treatment, 175(2), 339-351.

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Histological Evaluation of Mouse Pancreas

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Mouse pancreas was harvested and fixed in 10% neutral buffered formalin overnight at 4°C. Paraffin sections were embedded, sectioned, and hematoxylin and eosin (H&E)–stained by the UT Southwestern (UTSW) Molecular Pathology Core. Paraffin-embedded pancreas sections were deparaffinized in xylene and rehydrated through an ethanol gradient (100, 95, 70, and 50%). Antigen retrieval was performed for 30 min in citric acid–based antigen unmasking solution (Vector Laboratories) before immunofluorescence. H&E-stained pancreas was graded on an equal weight score (from 0 to 3) for edema (0 = absent; 1 = focally increased between lobules; 2 = diffusely increased; 3 = acini disrupted and separated), inflammatory infiltration (0 = absent; 1 = in ducts, around ductal margins; 2 = in the parenchyma, <50% of the lobules; 3 = in the parenchyma, >50% of the lobules), necrosis (0 = absent; 1 = periductal necrosis, <5% of cells; 2 = focal necrosis, 5 to 20% of cells; 3 = diffuse parenchymal necrosis, 20 to 50% of cells), and total severity score (sum of edema, inflammatory infiltrate, and necrosis scores) as previously described (31 (link)). Five different fields per mouse pancreas were analyzed in a blinded fashion. Images were acquired with a Zeiss Axio Scan.Z1 slide scanner.

Hernandez G., Luo T., Javed T.A., Wen L., Kalwat M.A., Vale K., Ammouri F., Husain S.Z., Kliewer S.A, & Mangelsdorf D.J. (2020). Pancreatitis is an FGF21-deficient state that is corrected by replacement therapy. Science translational medicine, 12(525), eaay5186.

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Immunohistochemical Staining of Paraffin-Embedded Tissue

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Formalin-fixed paraffin-embedded sections (4 μm) of omentum samples or co-cultures gels were heated for 1 hr at 60°C and then submerged twice in xylene for 5 min. Immunohistochemistry was performed using a Vectastain ABC Kit (Elite), as per the manufacturer's instructions. Slides were rehydrated and heated-antigen retrieval was performed using citric acid-based antigen unmasking solution (Vector Laboratories) and heated in a 2100 antigen-retriever (Aptum Biologics). Endogenous peroxidase activity was blocked using 3% H2O2 in PBS and sections were then blocked with 5% BSA. The primary antibody was added in antibody diluent (Zytomed) and covered at room temperature for 1 hr. Slides were incubated with a biotinylated secondary antibody (Vector) for 30 minutes. Subsequent steps were carried out according to the protocol included with the Vectastain Elite ABC HRP kit, after which the slides were incubated with DAB solution made using Sigmafast DAB tablets (Sigma-Aldrich). Finally, slides were counterstained in 50% Gill’s hematoxylin I, washed with water and dehydrated. After leaving to dry, coverslips were affixed using a drop of DPX mountant (Sigma-Aldrich).

Malacrida B., Nichols S., Maniati E., Jones R., Delanie-Smith R., Roozitalab R., Tyler E.J., Thomas M., Boot G., Mackerodt J., Lockley M., Knight M.M., Balkwill F.R, & Pearce O.M. (2021). A human multi-cellular model shows how platelets drive production of diseased extracellular matrix and tissue invasion. iScience, 24(6), 102676.

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Immunohistochemistry of Pancreatic Tissues

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Paraffin embedded pancreas sections were deparaffinized in xylene and rehydrated through an ethanol gradient (100%, 95%, 70% and 50%). Antigen retrieval was performed for 30 minutes in citric acid based antigen unmasking solution (Vector Laboratories). Sections were incubated for 1 hour in blocking buffer (1% BSA and 5% normal donkey serum). Where indicated, sections were incubated overnight at 4°C with primary antibodies to amylase (Santa Cruz), TdTomato (Rockland) or cytokeratin 19 (DSHB Cat# TROMA-III, RRID: AB 2133570). Sections were washed in PBS 3 times for 5 minutes followed by incubation for 1 hour at RT with Alexa-fluor conjugated secondary antibodies against the primary antibody’s host species. Sections were washed in PBS 3 times for 5 minutes and coverslipped using Vectashield with DAPI (Vector Laboratories). Images were acquired with a Zeiss Axioscan Z1 slide scanner.

Coate K.C., Hernandez G., Thorne C.A., Sun S., Le T.D., Vale K., Kliewer S.A, & Mangelsdorf D.J. (2017). FGF21 is an Exocrine Pancreas Secretagogue. Cell metabolism, 25(2), 472-480.

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Immunofluorescence Staining of CD105 and Insulin

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Cells were cultured to 70–80% confluence and dissociated into a single-cell suspension using TrypLE as described [33 (link)]. Cells and pancreatic tissue were then fixed in 10% cold formalin, prepared in a paraffin block, and sectioned. Antigen retrieval was performed using a citric acid-based antigen unmasking solution (Vector, pH 6.0). Sections were treated with protein block (Biogenex, Fremont, CA) to reduce background signal, followed by incubation with mouse anti-CD105 antibody (ready to use; Biogenex) and ALEXA 488-conjugated goat anti-mouse IgG antibody (1:200 dilution; ThermoFisher). Guinea pig anti-insulin (ThermoFisher) and ALEXA 647-conjugated goat anti-Guinea pig IgG antibodies (1:200 dilution; ThermoFisher) were used for pancreatic tissue staining only. Fluoroshield™ containing DAPI (Sigma Aldrich St. Louis, MO) was used to stain nuclei. Image acquisition was done using an Observer Z1 microscope (Carl Zeiss), with the objective lens set at 20×. Image processing was done using the Zen 2.0 software.

Khiatah B., Qi M., Du W., T-Chen K., van Megen K.M., Perez R.G., Isenberg J.S., Kandeel F., Roep B.O., Ku H.T, & Al-Abdullah I.H. (2019). Intra-pancreatic tissue-derived mesenchymal stromal cells: a promising therapeutic potential with anti-inflammatory and pro-angiogenic profiles. Stem Cell Research & Therapy, 10, 322.

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Immunohistochemical Analysis of ST6GAL1 in Rectal Adenocarcinoma

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Human rectal adenocarcinoma TMA tissue sections were deparaffinized and rehydrated by washing in xylene and ethanol. Slides were placed in boiling Citric Acid Based antigen unmasking solution (Vector). The slides were washed, covered in BLOXALL (Vector Labs) for 5 min and 0.5 % Triton-X in PBS for 25 min. Slides were blocked with 2.5 % horse serum and incubated with primary ST6GAL1 antibody 1:100 overnight (R&D Systems, #AF5924). ImmPRESS horse anti-goat immunoglobulin G (IgG, Vector Labs) was used as secondary, with Vector Labs NovaRED peroxidase substrate solution used for primary stain for two minutes. TMA slides were counterstained with hematoxylin for 30 s, dehydrated and mounted with coverslips.

Smithson M., Diffalha S.A., Irwin R.K., Williams G., McLeod M.C., Somasundaram V., Bellis S.L, & Hardiman K.M. (2024). ST6GAL1 is associated with poor response to chemoradiation in rectal cancer. Neoplasia (New York, N.Y.), 51, 100984.

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Immunohistochemical Localization of GPER

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The localization of GPER in the initial segment (IS), caput, corpus and cauda tissues was investigated by immunohistochemistry using a protocol similar to one previously described [3 (link)]. Tissue was paraffin embedded and sectioned at a thickness of 5 μm. Antigen retrieval was performed by submerging slides in a citric acid based antigen unmasking solution (Vector Laboratories, Inc., Burlingame CA) in Coplin jars and steam heated to 93 °C for 5 min after which they were allowed to cool to room temperature. Endogenous peroxidase activity was blocked by incubation in 0.3 % hydrogen peroxide in methanol for 30 minutes. After a blocking step, tissues were incubated for two hours at room temperature with rabbit anti-human GPR30 (1:2500; sc-48525; Santa Cruz Biotechnology). Following primary antibody incubation, sections were incubated with goat anti-rabbit biotinylated secondary antibody followed by an avidin–biotin horseradish peroxidase complex (Vector Laboratories). Immunostaining was visualized using NovaRed chromagen (Vector Laboratories). Pre-absorbed primary antibodies were substituted for primary antibody as a negative control; all other steps were identical including exposure times with NovaRed. All slides were counterstained with Immunomaster Hematoxylin and evaluated by light microscopy.

Martínez-Traverso G.B, & Pearl C.A. (2015). Immunolocalization of G protein-coupled estrogen receptor in the rat epididymis. Reproductive Biology and Endocrinology : RB&E, 13, 48.

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Immunohistochemical Analysis of Tumors

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Tumor samples were formalin-fixed and paraffin-embedded. 5 μm-thick sections were prepared. Immunohistochemical staining was used to assess for PDGFRα (1:250, Cell Signaling #3174) and CD31 (1:100, Cell Signaling #77699). Citric acid-based antigen unmasking solution (Vector Lab) was used. PBS with 0·3% Tween and 5% normal horse serum (Vector Lab) was used for blocking and as diluent for the primary and secondary antibodies. Slides were incubated in primary antibodies overnight and biotinylated secondary antibodies (1:200, Vector Lab #BA-1000) for 1 h. Expression was visualized with VECTASTAIN Elite ABC Reagent (Vector Lab) and 3,3′-Diaminobenzidine (DAB) solution (Vector Lab). Slides were counterstained with Mayer's hematoxylin (Sigma). For each mouse, four representative images were taken of the tumor if it occupied the majority of the slide (for PDGFRα staining: n = 19 for isotype, n = 19 for 1E10Fc, n = 20 for isotype + RT, and n = 18 for 1E10Fc + RT; for CD31 staining: n = 19 for isotype, n = 20 for 1E10Fc, n = 20 for isotype + RT, and n = 19 for 1E10Fc + RT). Each of the four images was quantified for fractional area positive for staining using ImageJ [15 (link)]. The four values were averaged for each mouse, and the averages were compiled for each treatment group. Experimenters were blinded to the treatment groups for staining and analysis.

Song E.J., Ashcraft K.A., Lowery C.D., Mowery Y.M., Luo L., Ma Y., Campos L.D., Cardona D.M., Stancato L, & Kirsch D.G. (2019). Investigating a chimeric anti-mouse PDGFRα antibody as a radiosensitizer in primary mouse sarcomas. EBioMedicine, 40, 224-230.

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