immunohistochemical staining of biomarkers in brain tumor neurosphere sections

Immunohistochemical Staining of Biomarkers in Brain Tumor Neurosphere Sections

First, melt the paraffin by incubating the slides in a metal rack at 60 degrees Celsius for 20 minutes. Then deparaffinized the slides via incubation in a rehydration train at room temperature according to the Tex protocol. Store the slides in tap water to prevent nonspecific antibody binding. After this, incubate the slides in citrate buffer at 125 degrees Celsius for 30 seconds at 90 degrees Celsius for 10 seconds. Allow the slides to cool before rinsing them five times with distilled water.Next, incubate the slides at room temperature in 0.3% hydrogen peroxide for 20 minutes. Then, wash the slides for five minutes three times in washing buffer with gentle agitation. Incubate the slides overnight in a humidified chamber set to 4 degrees Celsius with diluted primary antibody. After this, wash the slides three times for five minutes with gentle agitation.Next, incubate the slides in diluted biotinylated secondary antibody at room temperature for an hour. Wash the slides three times for five minutes in washing buffer with gentle agitation. Then, incubate the slides in avidin biotinylated horseradish peroxidase complex at room temperature in the dark for an hour. Repeat the wash as previously described. After this, incubate the slides with dab hydrogen peroxide substrate bands for two to five minutes at room temperature.Rinse the slides with tap water, and dip the slides in hematoxylin solution to counterstain them. Then rinse the slides thoroughly in tap water until the water runs clear. Dehydrate the slides according to the Tex protocol. Finally, coverslip the slides with a xylene-based mounting medium and allow them to dry on a flat surface at room temperature.

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1. Immunohistochemistry (IHC) of Paraffin-embedded neurospheres (NS)<ol><li>Melt paraffin by placing the slides in a metal rack and incubating them at 60 &#176;C for 20 min.</li><li>Deparaffinize the slides by incubation in a rehydration train at room temperature (RT): 3x xylene for 5 min, 100% 2x ethanol for 2 min, 95% 2x ethanol for 2 min, 70% 1x ethanol for 2 min, and 1x deionized water for 2 min. Hereon, keep the slides in tap water until antigen retrieval, as drying out will cause non-specific antibody binding.</li><li>Incubate the slides in citrate buffer (10 mM citric acid, 0.05% non-ionic detergent, pH 6) at 125 &#176;C for 30 s and at 90 &#176;C for 10 s. Let them cool down, then rinse the slides 5 times with distilled water.</li><li>Incubate slides at RT in 0.3% H2O2&#160;for 30 min.</li><li>Wash the slides 3 times in washing buffer (0.025% detergent in Tris-buffered saline [TBS]) for 5 min with gentle agitation.</li><li>Incubate the slides at RT with blocking solution (10% horse serum, 0.1% bovine serum albumin [BSA] in TBS) for 30 min.</li><li>Incubate the slides overnight at 4 &#176;C in a humidified chamber with primary antibody prepared in dilution solution (0.1% BSA in TBS). Wash the slides 3 times in washing buffer for 5 min with gentle agitation.</li><li>Incubate the slides at RT for 1 h with a biotinylated secondary antibody prepared in dilution solution. Wash the slides 3 times in washing buffer for 5 min with gentle agitation.</li><li>Incubate the slides at RT in the dark for 30 min with avidin-biotinylated horseradish peroxidase complex. Wash the slides 3 times in washing buffer for 5 min with gentle agitation.</li><li>Incubate the slides with 3,3&#8242;-Diaminobenzidine (DAB)-H2O2&#160;substrate brands here for 2-5 min at RT.</li><li>Rinse 3 times with tap water. Dip (1-2 s) the slides in hematoxylin solution to counterstain them, then rinse them thoroughly in tap water until clear.</li><li>Dehydrate the slides as follows: incubate in distilled water for 2 min, dip 3 times in 80% ethanol, incubate in 95% ethanol 2 times for 2 min each, incubate in 100% ethanol 2 times for 2 min each, and finally in xylene 3 times for 3 min each.</li><li>Coverslip the slides with a xylene-based mounting medium and let them dry on a flat surface at RT until they are ready for imaging.&#160;</li></ol>NOTE: If performing DAB staining, the slides can be stored at RT. However, if immunofluorescence staining is performed, slides should be stored in the dark at -20 &#176;C to reduce photo-bleaching.

<figure class='table' style='width:645pt;'><table class='ck-table-resized'><colgroup><col style='width:36.59%;'><col style='width:23.88%;'><col style='width:22.64%;'><col style='width:16.89%;'></colgroup><tbody><tr><td style='height:15.0pt;'>Rabbit polyclonal anti-ATRX,</td><td>Santa Cruz Biotechnology</td><td>sc-15408</td><td>IHC, 1:250 dilution</td></tr><tr><td style='height:15.0pt;'>Rabbit polyclonal anti-Ki-67</td><td>Abcam</td><td>Ab15580</td><td>IHC, 1:1000 dilution</td></tr><tr><td style='height:15.0pt;'>Rabbit polyclonal anti-OLIG2</td><td>Millipore</td><td>AB9610</td><td>IHC, 1:500 dilution</td></tr><tr><td style='height:15.0pt;'>Goat polyclonal anti-rabbit biotin-conjugated</td><td>Dako</td><td>E0432</td><td>IHC, 1:1000 dilution</td></tr><tr><td style='height:15.0pt;'>Vectastain Elite ABC HRP kit</td><td>Vector Laboratories Inc</td><td>PK-6100</td><td>&#160;</td></tr><tr><td style='height:15.0pt;'>BETAZOID DAB CHROMOGEN KIT</td><td>Biocare medical</td><td>BDB2004 L/price till 12/18</td><td>&#160;</td></tr></tbody></table></figure>

<a href='https://app.jove.com/v/58931/evaluation-biomarkers-glioma-immunohistochemistry-on-paraffin'>Source: N&#250;&#241;ez, F. J., et al. Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures.&#160;J. Vis. Exp. (2019).</a>This video demonstrates an immunohistochemical technique for labeling tumor biomarkers in paraffin-embedded brain tumor neurospheres. The neurosphere sections undergo paraffin removal and rehydration, followed by treatment to expose biomarker sites. Primary and secondary antibodies are then applied to label these biomarkers. The secondary antibodies are bound by avidin-conjugated peroxidase enzymes, which oxidize chromogenic substrates to indicate biomarker locations. Finally, nuclear counterstaining and slide preparation are completed for microscopic analysis.

Source: N&#250;&#241;ez, F. J., et al. Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere ...

Take a slide with paraffin-embedded sections of brain tumor neurospheres, clusters of neural stem cells exhibiting tumor-specific nuclear protein biomarkers.Heat to melt paraffin, remove it using xylene, and rehydrate the tissue using decreasing alcohol concentrations.Heat in an acidic buffer containing detergent to unmask epitopes on the biomarkers and permeabilize cellular membranes.Rinse, then treat with an oxidizing agent to inactivate endogenous peroxidase enzymes, preventing non-specific signals.Apply a blocking solution to mask non-specific binding sites.Incubate with primary antibodies targeting the biomarker epitopes.Rinse, then treat with biotin-conjugated secondary antibodies that bind to the primary antibodies.Rinse and incubate with a peroxidase enzyme-conjugated avidin, which binds to biotin.&#160;Add a chromogenic substrate and an oxidizing agent.Peroxidase utilizes the agent to convert the substrate into colored precipitates, labeling the biomarker location.Counterstain the nuclei, dehydrate the tissue in alcohol, and mount the slide for imaging the biomarker distribution.

6 Views. Immunohistochemical Staining of Biomarkers in Brain Tumor Neurosphere Sections

Video: Immunohistochemical Staining of Biomarkers in Brain Tumor Neurosphere Sections - Experiment

Synthetic Antigen Controls for Immunohistochemistry

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 &#176;C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities.
This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.

This work documents a simple method to create synthetic antigen controls for immunohistochemistry. The technique is adaptable to a variety of antigens in a wide range of concentrations. The samples provide a reference with which to assess intra- and inter-assay performance and reproducibility.

Immunohistochemical Staining of Biomarkers in Brain Tumor Neurosphere Sections

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Immunohistochemical Staining of Biomarkers in Brain Tumor Neurosphere Sections