immunohistochemistry to study the relationship between macrophages and infiltrated immune cells

Immunohistochemistry to Study the Relationship Between Macrophages and Infiltrated Immune Cells

For antigen retrieval, transfer the rack into a glass container with 200 microliters of 95 degrees Celsius, 10 millimolar per liter, citrate buffer for 30 minutes. At the end of the incubation, allow the slides to cool to room temperature for 20 minutes before rinsing with running water for five minutes.
To remove the endogenous peroxidase activity, first, use a hydrophobic marker to draw a circle around each tissue sample, before incubating the samples in 200 microliters of 3% hydrogen peroxide for 10 minutes, followed by three washes in 200 microliters of Tris-buffered saline solution or TBSS per wash. After the last wash, dab each slide gently to remove any excess buffer, and add 200 microliters of the primary antibody cocktail of interest and a coverslip to each slide for a one-hour incubation in a humid chamber at room temperature.
At the end of the incubation, discard the coverslips and rinse the slides with three two-minute washes in fresh TBSS. After removing the excess buffer, add 200 microliters of an appropriate biotinylated secondary antibody solution and a coverslip to each slide for a 45-minute incubation in the humid chamber at room temperature. After discarding the coverslips, rinse the slides with TBSS, as demonstrated. Remove any excess buffer.
Enable the sections with 200 microliters of DAB substrate buffer for 10 minutes at room temperature. At the end of the incubation, wash the slides with TBSS and running tap water for 10 minutes, before counter-staining with hematoxylin for three minutes. Then, add 70 microliters of an appropriate mounting medium to each slide, and slowly tip a glass coverslip onto the mounting medium to avoid creating bubbles as the coverslip is lowered into place.
For histopathologic analysis, place the slide onto the stage of a light microscope and assess the vascular architecture of the first tissue section. Then, quantify the number of macrophages relative to the number of lymphocytes per section.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Histopathological Preparation, and Processing After Obtaining Temporal Artery Biopsy
NOTE:&#160;The temporal artery biopsy (TAB) is performed under local anesthesia and sterile conditions by a certified surgeon. After surgically obtaining a 3 cm arterial section on the more affected side, the specimen is fixed in formalin immediately for 24 h, divided into 3 to 4 mm sections, and embedded in paraffin, paying careful attention to the proper alignment of tissue in the block which is then stored at room temperature. Specimen selection is performed after verifying the medical records. The diagnosis is based on the American College of Rheumatology 1990 criteria which requires that subjects fulfill 3 of 5 domains: age &#62;50 years, new headache, temporal artery tenderness, an erythrocyte sedimentation rate &#62;50 mm/hour by Westergren method, and an abnormal TAB.&#160;For safety, protective gloves must be worn at all times when handling specimens, chemicals, and antibodies.
<ol>
	<li>From the embedded blocks, cut 4-5 &#181;m thick sections using a microtome. and stain with Hematoxylin and Eosin (H&#38;E). Use a control section of selected normal tissue for quality assurance.</li>
	<li>Float cut sections on a warm water bath heated to 40 &#176;C to remove wrinkles and pick them up with a coated glass microscopic slide.&#160; Place the glass slides on a warm plate in a 60 &#176;C oven for 60 minutes to facilitate drying and adherence of the section to the slide.</li>
	<li>Place the glass slides on a warm plate in a 60 &#176;C oven for 60 minutes to facilitate drying and adherence of the section to the slide.</li>
	<li>Mount 3 sections on microscope slides.</li>
	<li>Place slides in a vertical rack and dry overnight at 37 &#176;C for 24 h.</li>
	<li>Place slides in a container and store at 4 &#176;C.</li>
</ol>
2. Immunohistochemical Preparation, Dewaxing,&#160;Antigen Retrieval and Staining
<ol>
	<li>Load slides into a slide rack. Run the rack through dishes as follows: Twice in 100% xylene for 5 minutes with 10 seconds of gentle agitation every 30 seconds, once in 100% ethanol with 10 seconds agitation, once in 90% ethanol with 10 seconds agitation, once in 70% ethanol with 10 seconds agitation, and twice in double distilled H2O with 10 seconds agitation.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Handling of xylene and acetone should be done in ventilated hoods to avoid inhalation and respiratory toxicity.</li>
	<li>Retrieve the antigen by transferring the rack into a glass container with 200 mL of 10 mmol/L, pH 6.0 citrate buffer prewarmed to 95 &#176;C. Set the timer for 30 minutes in the water bath, then cool at room temperature for 20 minutes then rinse with running water for 5 minutes.</li>
	<li>Remove endogenous peroxidase activity by incubating in 200 mL of 3% hydrogen peroxide for 10 minutes. Wash slides 3 times in 200 &#181;L of Tris-buffered saline solution (TBSS).</li>
	<li>Remove the slides from the rack and dab each one gently to wipe out excess buffer. For staining, add 200 &#181;L of the following primary antibodies, add coverslips on slides, and incubate in a humid chamber for 1 hour at room temperature: 
	Anti-folate receptor beta (FRB) antibody diluted 1:800&#160;&#160; 
	Monoclonal mouse anti-human CD68, 1:200 dilution&#160;&#160; 
	Polyclonal rabbit anti-CD3 1:100 dilution 
	NOTE: Formalin-fixed paraffin-embedded sections of the placenta were also processed as outlined in Steps 2.1 to 2.9, and incubated with anti-FRB18, and used as a positive control. Staining results were obtained by finding the optimal antibody concentration and was performed by titrating the antibody in double dilutions (e.g., 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600).</li>
	<li>Remove the coverslip after incubation and discard. Rinse slides 3 times for 2 minutes each with TBSS, drain, and wipe excess buffer.</li>
	<li>Add 200 &#181;L of secondary antibody solution containing biotinylated Anti-Rabbit/Mouse secondary antibody to react with unconjugated primary antibody bound to tissue antigen. Place coverslips on slides and incubate in a humid chamber for 45 minutes at room temperature.</li>
	<li>Remove the coverslip after incubation and discard. Rinse slides 3 times for 2 minutes each with TBS, drain, and wipe excess buffer.</li>
	<li>Add 200 &#181;L of diaminobenzidine (DAB)+substrate buffer (Imidazole HCl) -chromogen system and incubate for 10 minutes to visualize the staining pattern. Wash with TBSS then in running tap water for 10 minutes.</li>
	<li>Counterstain with hematoxylin for 3 minutes.</li>
	<li>Mount the slides by applying 70 &#181;L of mounting medium to the surface of the slide. Slowly tip the coverslip onto the mounting medium and avoid creating bubbles as you lower it into place. Wait 24 hours to dry.&#160;</li>
</ol>
3. Histopathologic and Immunohistochemical (IHC) Analysis
Examine the H&#38;E and IHC stained sections from GCA-positive and normal subjects using a light microscope.
<ol>
	<li>For the H&#38;E stained sections
	<ol>
		<li>Assess the vascular architecture and identify endothelial cells in the tunica intima, smooth muscle cells in the tunica media, and fibroblasts and vasa vasorum in the tunica adventitia.</li>
		<li>Identify GCA features, which include lymphocyte and macrophage/histiocyte infiltration, cellular hyperplasia, and internal elastic lamina degradation.</li>
		<li>Analyze the lymphohistiocytic infiltrate by identifying and quantifying the number of macrophages relative to the lymphocytes. Assess the extent of internal elastic lamina disruption and hyperplastic changes in resident cells and compare them with controls.</li>
	</ol>
	</li>
	<li>For the IHC stained sections
	<ol>
		<li>Examine the staining pattern of FRB, taking note of its expression by a particular type or types of cells and its distribution within the vascular layers and its relationship to the total immune infiltrate, the total macrophage marker, anti-CD68, and the pan lymphocyte marker anti CD3.</li>
		<li>Quantify the expression of FRB, CD68, and CD3 cells by counting the positively stained cells on 10 multiple randomly selected high power fields (hpf) and record the means.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE:&#160;Identification of these normal and pathologic cells and structures was performed by a certified cardiovascular pathologist and rheumatologist, and the results were recorded for all cases.</li>
		<li>Capture representative images using a digital camera. 
		NOTE:&#160;After these steps, remaining aliquots may be stored at -80 &#176;C.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Microtome</td>
			<td>Reichert-Jung</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plain and frosted microscope slides and cover slips</td>
			<td>Fisher Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical rack</td>
			<td>Fisher Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus BX60 microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone in distilled water</td>
			<td></td>
			<td></td>
			<td>concentrations 100%, 90%, 70%</td>
		</tr>
		<tr>
			<td>Tris-buffered saline solution (TBS)&#160;</td>
			<td>Dako Wash BufferS3006</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3% hydrogen peroxide in TBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diaminobenzidine (DAB)</td>
			<td>Sigma Fast Tablet set</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemical Permount&#160; Mounting Medium</td>
			<td>Fisher Scientific&#160;</td>
			<td>SP-15-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Harleco Gill&#39;s III Hematoxylin</td>
			<td>Fiisher Scientific 23-750-019</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Harleco Eosin Y 1% Alcoholic Stock Solution</td>
			<td>Fisher Scientific&#160;</td>
			<td>23-749-977</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM citric acid monohydrate (pH 6.0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal rabbit anti-folate receptor beta&#160;</td>
			<td>Manohar Ratnam</td>
			<td></td>
			<td>optimized at 1:800</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-human CD68</td>
			<td>Dako&#160;</td>
			<td>M071801</td>
			<td>optimized at 1:200</td>
		</tr>
		<tr>
			<td>Polyclonal rabbit anti-CD3</td>
			<td>Agilent Dako</td>
			<td>A0452</td>
			<td>optimized at 1:100</td>
		</tr>
		<tr>
			<td>Polymer goat anti- rabbit/mouse secondary antibody</td>
			<td>Dako</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Placental tissue</td>
			<td></td>
			<td></td>
			<td>&#160;for FRB positive control</td>
		</tr>
	</tbody>
</table>

Source: Albano-Aluquin, S. et al., <a href="https://app.jove.com/v/58713">An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis.</a> J. Vis. Exp.&#160;(2019)
This video demonstrates a technique to quantify the relative ratio of macrophages among the total infiltrated immune cell populations in temporal artery biopsy. The immunohistochemical staining of a tissue section affected by autoimmune disease selectively stains the immune cells with specific receptors to visually differentiate them from the rest of the cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Histopathological Preparation, ...

Begin with a slide containing formalin-fixed, deparaffinized, and rehydrated temporal artery biopsy sections with infiltrated immune cells, including lymphocytes and macrophages.
Activated macrophages express folate receptor beta or FR-&#946; &#8212; a disease-related marker, differentiating the macrophages from other immune cells.
Transfer the slide into a pre-warmed buffer to retrieve surface receptors.
Treat the section with hydrogen peroxide, blocking intracellular peroxidase activity.
Introduce a mix of primary antibodies targeting CD3, CD68, and FR-&#946;, which bind to immune cells expressing these proteins.
Add biotinylated secondary antibodies, interacting with primary antibodies, and wash.
Overlay with streptavidin-conjugated enzymes that bind with biotin.
Treat with a chromogenic substrate that interacts with enzymes, producing localized brown precipitates.
Introduce a counterstain &#8212; hematoxylin, staining the nuclei blue.
Microscopically identify small, round, brown-stained cells as CD3-expressing lymphocytes and large, irregular, brown-stained cells as CD68 and FR-&#946;-expressing macrophages.
Quantify macrophages in the section to find their ratio amongst the total infiltrated immune cells.

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视频: 免疫组化研究巨噬细胞与浸润免疫细胞之间的关系 - 实验

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

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Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

免疫与感染

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Immunohistochemistry to Study the Relationship Between Macrophages and Infiltrated Immune Cells

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Immunohistochemistry to Study the Relationship Between Macrophages and Infiltrated Immune Cells