Antigen Masking During Fixation and Embedding, Dissected

Antigen masking in routinely processed tissue is a poorly understood process caused by multiple factors. We sought to dissect the effect on antigenicity of each step of processing by using frozen sections as proxies of the whole tissue. An equivalent extent of antigen masking occurs across variable fixation times at room temperature. Most antigens benefit from longer fixation times (>24 hr) for optimal detection after antigen retrieval (AR; for example, Ki-67, bcl-2, ER). The transfer to a graded alcohol series results in an enhanced staining effect, reproduced by treating the sections with detergents, possibly because of a better access of the polymeric immunohistochemical detection system to tissue structures. A second round of masking occurs upon entering the clearing agent, mostly at the paraffin embedding step. This may depend on the non-freezable water removal. AR fully reverses the masking due both to the fixation time and the paraffin embedding. AR itself destroys some epitopes which do not survive routine processing. Processed frozen sections are a tool to investigate fixation and processing requirements for antigens in routine specimens.

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Application of Heat-induced Antigen Retrieval to Aldehyde-fixed Fresh Frozen Sections

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.4a6579.2005 ◽

2005 ◽

Vol 53 (11) ◽

pp. 1421-1432 ◽

Cited By ~ 31

Author(s):

Shuji Yamashita ◽

Yasunori Okada

Keyword(s):

Room Temperature ◽

Glucocorticoid Receptors ◽

Estrogen Receptor Α ◽

Antigen Retrieval ◽

Frozen Sections ◽

New Approach ◽

Cacodylate Buffer ◽

Ligand Free ◽

Fresh Frozen ◽

Mouse Hepatocytes

We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl2 for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl2, 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor α (ERα) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERα immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl2 at 40C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.

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Epidermal growth factor receptor expression correlates with poor survival in gastric adenocarcinoma from Mexican patients: a multivariate analysis using a standardized immunohistochemical detection system

Modern Pathology ◽

10.1038/modpathol.3800085 ◽

2004 ◽

Vol 17 (5) ◽

pp. 579-587 ◽

Cited By ~ 58

Author(s):

Armando Gamboa-Dominguez ◽

Claudia Dominguez-Fonseca ◽

Leticia Quintanilla-Martinez ◽

Edgardo Reyes-Gutierrez ◽

Dan Green ◽

...

Keyword(s):

Epidermal Growth Factor Receptor ◽

Multivariate Analysis ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Detection System ◽

Growth Factor Receptor ◽

Receptor Expression ◽

Immunohistochemical Detection ◽

Poor Survival ◽

Epidermal Growth

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The Contribution of Bifunctional SkipDewax Pretreatment Solution, Rabbit Monoclonal Antibodies, and Polymer Detection Systems in Immunohistochemistry

Archives of Pathology & Laboratory Medicine ◽

10.5858/2007-131-1047-tcobsp ◽

2007 ◽

Vol 131 (7) ◽

pp. 1047-1055

Author(s):

Sze Chuen Cesar Wong ◽

John K. C. Chan ◽

Elena S. F. Lo ◽

Amanda K. C. Chan ◽

Manson C. K. Wong ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Colorectal Carcinoma ◽

Diagnostic Accuracy ◽

Poor Quality ◽

Antigen Retrieval ◽

Ki 67 ◽

Detection Systems ◽

Edta Solution ◽

Endogenous Biotin ◽

Pretreatment Solution

Abstract Context.—In immunohistochemistry, nonstandardized antigen retrieval protocols and fluids, poor-quality antibodies, and the presence of endogenous biotin frequently lead to incorrect results. Recently, advanced reagents including bifunctional SkipDewax pretreatment solution (BSPS), rabbit monoclonal (RM) antibodies, and biotin-free polymer detection systems (PDSs) have been developed, which, it is claimed, resolve these problems. Objectives.—To determine whether BSPS, RM antibodies, and biotin-free PDSs improve the accuracy of immunohistochemistry; to optimize a new protocol consisting of a combination of BSPS, RM antibodies, and PDSs; and to compare it with a conventional protocol. Design.—The efficacies of BSPS, RM antibodies, and PDSs were compared with those of their respective conventional reagents using multitissue spring-roll sections. The new protocol was compared with a conventional protocol using Ki-67 immunostaining of 49 colorectal carcinoma specimens. Results.—For antigen retrieval, BSPS resulted in similar or better tissue staining than an EDTA solution, but the efficacy of BSPS decreased when it was reused. Most RM antibodies resulted in a greater proportion of positive cells than the corresponding non-RM antibodies, which did not produce satisfactory results in the absence of antigen retrieval. The PDSs Bond, ChemMate, and SuperPicture resulted in a high percentage of positive cells, good staining intensities, and low backgrounds. Other PDSs, except that from Ventana, resulted in high backgrounds and false positivity. The new combined protocol resulted in better Ki-67 staining than the conventional assay. Conclusions.—Bifunctional SkipDewax pretreatment solution, RM antibodies, and PDSs improve staining quality and diagnostic accuracy of immunohistochemistry assays and provide a foundation for standardization.

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Artifacts associated with mithramycin fluorescence in the clinical detection and quantitation of aneuploidy by flow cytometry.

Journal of Histochemistry & Cytochemistry ◽

10.1177/30.4.6460801 ◽

1982 ◽

Vol 30 (4) ◽

pp. 317-322 ◽

Cited By ~ 8

Author(s):

R E Cunningham ◽

K S Skramstad ◽

A E Newburger ◽

S E Shackney

Keyword(s):

Flow Cytometry ◽

Room Temperature ◽

Human Melanoma ◽

Time Dependent ◽

Clinical Samples ◽

Fixation Time ◽

C Cells ◽

Temperature Dependent ◽

Clinical Detection

Ethanol-fixed cells stored at 4 degrees C exhibit fixation time-dependent hyperchromatism in comparison with freshly fixed cells when stained with mithramycin and examined by flow cytometry. This hyperchromatism has been found to be temperature-dependent, developing fully within 72 hr at room temperature, and within 2 hr at 37 degrees C. Cells from normal donors that are stained with mithramycin exhibit spurious aneuploid peaks. These spurious aneuploid peaks can be eliminated by incubating ethanol-fixed cells at 37 degrees C for 2 hr prior to staining; true aneuploidy is not affected by this procedure. In rare instances, cytoplasmic fluorescence can be observed in mithramycin-stained cells. In addition, unexplained hypochromatism and hyperchromatism can be observed in some clinical samples, particularly in human melanoma. The effects of these unexplained staining artifacts can be minimized or eliminated by adopting strict criteria for the clinical detection of aneuploidy by flow cytometry.

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Immunohistochemical Detection of Proliferative Marker Ki-67 in Benign and Malignant Salivary Gland Tumors

The Journal of Contemporary Dental Practice ◽

10.5005/jp-journals-10024-2269 ◽

2018 ◽

Vol 19 (4) ◽

pp. 375-383

Author(s):

Smita Bussari ◽

Sindhu M Ganvir ◽

Manish Sarode ◽

Prabhakar A Jeergal ◽

Anjum Deshmukh ◽

...

Keyword(s):

Salivary Gland ◽

Salivary Gland Tumors ◽

Immunohistochemical Detection ◽

Ki 67 ◽

Proliferative Marker ◽

Malignant Salivary Gland Tumors

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A Rare Primary Tumor of the Thyroid Gland: A New Case of Leiomyoma and Literature Review

Clinical Medicine Insights Oncology ◽

10.1177/1179554918813535 ◽

2018 ◽

Vol 12 ◽

pp. 117955491881353

Author(s):

Yanling Zhang ◽

Heng Tang ◽

Huaiyuan Hu ◽

Xiang Yong

Keyword(s):

Thyroid Gland ◽

Tumor Cells ◽

Immunohistochemical Staining ◽

Smooth Muscle Actin ◽

Frozen Sections ◽

Ki 67 ◽

Eosinophilic Cytoplasm ◽

S 100 ◽

Painless Mass ◽

The Right

Primary leiomyomas of the thyroid are very rare. We here report a case of a 53-year-old woman with a painless mass at the right thyroid, revealed by physical examination. The patient underwent a lobectomy. Frozen sections showed a spindle cell tumor of the thyroid gland. The nuclei of some of the tumor cells were obviously enlarged and deeply stained. Pseudocapsule invasion was observed in small foci. Samples showed neither mitosis nor necrosis and the nature of the tumor was difficult to determine. Paraffin sections showed a well-circumscribed nodular composed of intersecting fascicles of spindled to slightly epithelioid cells with eosinophilic cytoplasm and blunt-ended, cigar-shaped nuclei. We observed no significant nuclear atypia, mitotsis, or necrosis. Immunohistochemical staining showed the tumor cells to be positive for α-smooth muscle actin and h-caldesmon but negative for TG, TTF1, PAX8, S-100, CT, CK, and CD34. The ki-67 index was very low (<1%). Primary thyroid leiomyoma is rare and difficult to diagnose using frozen sections. Diagnosis requires immunohistochemical staining. Leiomyoma may be mistaken for other thyroid tumors also characterized by spindle cells.

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Long-term surgical anaesthesia with isoflurane in human habituated Nile Crocodiles

Journal of the South African Veterinary Association ◽

10.4102/jsava.v88i0.1451 ◽

2017 ◽

Vol 88 ◽

Cited By ~ 1

Author(s):

George F. Stegmann ◽

Catherine J.A. Williams ◽

Craig Franklin ◽

Tobias Wang ◽

Michael Axelsson

Keyword(s):

Body Temperature ◽

Body Mass ◽

Room Temperature ◽

Environmental Temperature ◽

Recovery Period ◽

Physiological Data ◽

Surgical Incision ◽

Multiple Factors ◽

Physiological Sensors

A suitable long-term anaesthetic technique was required for implantation of physiological sensors and telemetric devices in sub-adult Nile crocodiles (Crocodylus niloticus) to allow the collection of physiological data. Five Nile crocodiles with a median body mass of 24 kg were used. After manual capture, they were blindfolded and 0.2 mL (1 mg/mL) medetomidine was administered intramuscularly in four of the animals which had an estimated body mass between 20 kg and 30 kg. One crocodile with an estimated body mass of 50 kg received 0.5 mL. For induction, 5 mL propofol (10 mg/mL) was injected intravenously into the occipital sinus. Additional doses were given when required to ensure adequate anaesthesia. Anaesthesia was maintained with 1.5% isoflurane. Ventilation was controlled. Local anaesthesia was administered for surgical incision and external placement of the radio transmitter. Medetomidine was antagonised with atipamezole at the end of surgery. Median heart rate during surgery was 22 beats/min, at extubation 32 beats per min and 30 beats per min the following day at the same body temperature as under anaesthesia. Median body temperature of the animals increased from 27.3 °C to 27.9 °C during anaesthesia, as room temperature increased from 24.5 °C to 29.0 °C during surgery. Anaesthesia was successfully induced with intramuscular medetomidine and intravenous propofol and was maintained with isoflurane for the placement of telemetric implants. Intraoperative analgesia was supplemented with lidocaine infiltration. Perioperative physiological parameters remained stable and within acceptable clinical limits. Multiple factors appear to influence these variables during the recovery period, including residual anaesthetic effects, environmental temperature and physical activity.

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MICA—A Highly Sensitive and Avidin-Free Immunohistochemical Detection System

Advances in Anatomic Pathology ◽

10.1097/00125480-200007060-00003 ◽

2000 ◽

Vol 7 (6) ◽

pp. 360-364 ◽

Cited By ~ 2

Author(s):

D. C. Mangham ◽

A. R. Bradwell ◽

P. G. Isaacson

Keyword(s):

Detection System ◽

Immunohistochemical Detection ◽

Highly Sensitive

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Simultaneous determination of mercury speciation in biological materials by GC/CVAFS after ethylation and room-temperature precollection

Clinical Chemistry ◽

10.1093/clinchem/40.4.602 ◽

1994 ◽

Vol 40 (4) ◽

pp. 602-607 ◽

Cited By ~ 106

Author(s):

L Liang ◽

N S Bloom ◽

M Horvat

Keyword(s):

Simultaneous Determination ◽

Room Temperature ◽

Detection System ◽

Inorganic Mercury ◽

Biological Materials ◽

Atomic Fluorescence Spectrometry ◽

Atomic Fluorescence ◽

Monomethyl Mercury ◽

Isothermal Gas

Abstract We developed a method for the simultaneous determination of monomethyl mercury (MMHg), inorganic mercury [Hg(II)], and total mercury (THg) in biological materials. A variety of biological materials can be digested in methanolic KOH solution. The MMHg and Hg(II) present are converted to volatile ethyl derivatives, methylethyl mercury and diethyl mercury, by an aqueous-phase ethylation reaction with sodium tetraethylborate. The ethyl derivatives are precollected onto a trapping column at room temperature, in case of disconnection with the separation/detection system, and then thermally desorbed into a packed isothermal gas chromatography (GC) column. Eluted organo-Hg compounds from the GC column are decomposed into Hg0, and detection is completed by cold vapor atomic fluorescence spectrometry (CVAFS). Pure standard solutions can be used for calibration. The sum of MMHg and Hg(II) obtained by this method equals the THg value obtained by digestion with HNO3 and H2SO4, reduction with SnCl2, and single-stage amalgamation/CVAFS for all biological materials studied. Absolute detection limits are 0.6 pg and 1.3 pg of Hg as MMHg and Hg(II), respectively, corresponding to 0.3 ng and 0.6 ng/g (wet) of sample.

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