The Antigen-Antibody Reaction in Immunohistochemistry

Montero, Claudio

doi:10.1177/002215540305100101

Cited by 73 publications

(50 citation statements)

References 28 publications

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“…P-gp-labeled capillary endothelial cells are marked by "e" and astroglial cells by "a." nochemical integrity of the antigen (Shi et al 1997;Werner et al 2000;MacIntyre 2001;Montero 2003). As previously described by Thiebaut et al (1989), the P-gp epitope reactive with C219 is very aldehyde sensitive, so that formaldehyde fixation may dramatically reduce its endothelial localization in brain tissue as compared with acetone-fixed sections.…”

Section: Discussionmentioning

confidence: 83%

Immunohistochemical Localization of P-glycoprotein in Rat Brain and Detection of Its Increased Expression by Seizures Are Sensitive to Fixation and Staining Variables

Volk

Potschka

Löscher

2005

J Histochem Cytochem.

103

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S U M M A R YThe MDR1 gene product, P-glycoprotein (P-gp), was shown to confer multidrug resistance to cancer cells, but its overexpression is also suggested to be involved in pharmacoresistance of epilepsy by acting as an energy-dependent drug-efflux pump in the blood-brain barrier (BBB). In normal brain tissue, P-gp is almost exclusively expressed by capillary endothelial cells (EC) of the BBB, whereas little or no expression is detected in other cell types. Increased P-gp expression was observed after seizures, but localization of this increase, i.e., within brain capillary EC or within parenchymal or perivascular astrocytes, which contribute to the BBB function, is controversial. To test whether these antithetic data arise from unusual properties of the antigen itself, we compared different immunohistochemical techniques and monoclonal or polyclonal antibodies to P-gp in normal rat brain and rat brain after kainate-induced seizures. Using acetone-fixed cryostat sections of snap-frozen tissue, strong P-gp labeling was detected in EC and, after seizures, in hippocampal neurons, but not in astrocytes. In contrast, EC and neuronal P-gp immunolabeling were not seen in paraformaldehyde-fixed sections, whereas both perivascular and parenchymal astrocytes exhibited strong P-gp labeling after seizures. The lack of P-gp labeling in EC by paraformaldehyde fixation, was reversed by treatment of the sections with acetate/ethanol. These experiments demonstrate that various fixation conditions have a striking effect on the immunohistochemical localization of P-gp in rat brain and detection of its increased expression by seizures. When data obtained from different immunohistochemical techniques are taken together, seizures seem to induce overexpression of P-gp in four different cell types, i.e., EC, perivascular astrocytes, parenchymal astrocytes, and neurons.

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Section: Discussionmentioning

confidence: 83%

Immunohistochemical Localization of P-glycoprotein in Rat Brain and Detection of Its Increased Expression by Seizures Are Sensitive to Fixation and Staining Variables

Volk

Potschka

Löscher

2005

J Histochem Cytochem.

103

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“…For example, nonstandardized, delayed, or excessive fixation of tissue specimen can result in differential expression of protein markers (39). Furthermore, certain epitopes may be masked, poorly retrieved, or absent in tumor samples even if a proven antibody is used (40). Lastly, an activated receptor may mask antibody-epitope recognition while in its phosphorylated state (41).…”

Section: Discussionmentioning

confidence: 99%

Enhanced Killing of Primary Ovarian Cancer by Retargeting Autologous Cytokine-Induced Killer Cells with Bispecific Antibodies: A Preclinical Study

Chan¹,

Hamilton²,

Cheung³

et al. 2006

Clinical Cancer Research

112

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Cr release assay of fresh ovarian cancer cells exposed to autologous CIK cells increased from 21.7 F 0.3% to 89.4 F 2.1% at an E:Tratio of100:1 (P < 0.001). Anti-NKG2D antibodies attenuated the CIK activity by 56.8% on primary cells (P < 0.001). In a xenograft severe combined immunodeficient mouse model, real-time tumor regression and progression was visualized using a noninvasive in vivo bioluminescence imaging system. Four hours after CIK cell injection, we were able to visualize CD8 + NKG2D + CIK cells infiltrating Her2-expressing cancer cells on fluorescence microscopy. Mice that underwent adoptive transfer of CIK cells redirected with BSAbxCA125 and BSAbxHer2 had significant reduction in tumor burden (P < 0.001and P < 0.001) and improvement in survival (P = 0.05 and P = 0.006) versus those treated with CIK cells alone. Bispecific antibodies significantly enhanced the cytotoxicity of CIK cells in primary ovarian cancer cells and in our in vivo mouse model. The mechanism of cytolysis seems to be mediated in part by the NKG2D receptor.

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“…The forces and bonds that participate in immune reactions are electrostatic charges, van der Waals forces, hydrogen bonds and hydrophobic interactions 32 . Therefore, the AR procedure, by using enzymatic methods or heating , can expose antigenic epitopes in order to enhance immunostaining in the tissue 1 , stabilizing the electrostatic forces that are necessary to bind the antigen and antibody 32 .…”

Section: Discussionmentioning

confidence: 99%

“…Immunohistochemistry (IHC) is an important technique for pathology diagnostics and investigative studies 1 . In the first step of IHC, tissue is fixed in a solution that usually contains formalin; however the use of this component is extensively discussed because many antigens interact with the chemical [1][2][3][4] .…”

Section: Introductionmentioning

confidence: 99%

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Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: Myosin-v assay

et al. 2010

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The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 o C, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.

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The Antigen-Antibody Reaction in Immunohistochemistry

Cited by 73 publications

References 28 publications

Immunohistochemical Localization of P-glycoprotein in Rat Brain and Detection of Its Increased Expression by Seizures Are Sensitive to Fixation and Staining Variables

Immunohistochemical Localization of P-glycoprotein in Rat Brain and Detection of Its Increased Expression by Seizures Are Sensitive to Fixation and Staining Variables

Enhanced Killing of Primary Ovarian Cancer by Retargeting Autologous Cytokine-Induced Killer Cells with Bispecific Antibodies: A Preclinical Study

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: Myosin-v assay

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