The authors thank Drs. Fahad Bafakih and Jim Mandell for acquisition and identification of patient samples, Garrett F. Beeghly for assistance with immunohistochemistry, and the Biorepository and Tissue Research Facility, the Cardiovascular Research Center Histology Core, and the Biomolecular Analysis Facility at the University of Virginia for assistance with sample acquisition, immunohistochemistry, and imaging.

This protocol identifies cellular components in formalin-fixed paraffin embedded (FFPE) samples, as is typical for banked clinical patient samples. Paraffin embedding allows for the best maintenance of cellular and tissue morphology as well as has better longevity of sections. The samples used for this analysis were accessed through the University of Virginia Biorepository and Tissue Research Facility. Patient samples were selected by a neuropathologist based on a definitive diagnosis of glioblastoma (astrocytoma, WHO gr...

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Our method proposed here is a quantitative approach to analyzing histological samples stained using traditional chromogenic immunohistochemistry. Current methodology for this type of analysis includes similar staining protocols followed by grading by independent pathologists. This method has been reliable, yet for a number of applications, a more precise understanding of the cellular make-up is required, such as better understanding of the heterogeneity associated with tumors and accurate recapitulation of tumors for in ...

Glioblastoma (GBM), the most common and malignant brain cancer, is characterized by highly diffuse invasion from the primary tumor bulk into the surrounding healthy brain parenchyma1,2. This diffuse invasion makes the tumor particularly difficult to resect fully, and the invading cancer cells that remain post-therapy is the most common reason for inevitable recurrence2,3,4. Previously, we found inhibiting the diffuse glioma cell invasion to be therapeutically beneficial5, however little is known about the complex mechanisms contributing to GBM invasion. The tumor microenvironment, or tissue surrounding the cancer, has been implicated in the progression of tumors in multiple cancers6,7. The glioblastoma tumor microenvironment, in particular, is relatively under-characterized and is uniquely complex, composing of multiple glial cells, such as astrocytes, microglia, and oligodendrocytes, as well as extracellular matrix, soluble factors, and biophysical factors. Experimentally, astrocytes and microglia have been shown to increase glioma progression and invasion8,9,10, but the composition of all glial cells in the native human brain microenvironment is unknown.
We previously showed microenvironmental components can predict patient survival by quantitatively analyzing cellular components of the glioblastoma microenvironment and incorporating our analyses into a proportional hazards model11. Here, we describe the quantitative analysis method for identifying the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations and ImageJ to analyze percent coverage of staining for each glial population. Assessing percent coverage creates a simple measurement for determining the morphological differences of cells, particularly those affected by interactions with cancer cells. Previous studies for quantifying histopathological staining use standard staining such as hematoxylin and eosin12 or Masson&#39;s trichrome13, which do not take advantage of the specificity of antibody-based immunohistochemistry staining. Our method was developed to directly quantify the glial populations within glioblastoma patient tumor resections, which we aim to use to elucidate the complex glioblastoma microenvironment.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Xylene</td>
			<td style="width:71px;">Fisher Chemical</td>
			<td style="width:119px;">X3P</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">High pH antigen unmasking solution</td>
			<td style="width:71px;">Vector Labs</td>
			<td>H-3301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TBS</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton-X</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">9002-93-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Horse serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-ALDH1L1&#160;</td>
			<td style="width:71px;">abcam&#160;</td>
			<td style="width:119px;">ab56777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Iba1&#160;</td>
			<td style="width:71px;">abcam&#160;</td>
			<td style="width:119px;">ab5076</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-Oligodendrocyte Specific Protein1&#160;</td>
			<td style="width:71px;">abcam&#160;</td>
			<td style="width:119px;">ab53041</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ImmPRESS anti-goat</td>
			<td style="width:71px;">Vector Labs</td>
			<td style="width:119px;">MP-7405</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ImmPRESS Universal (anti-mouse/rabbit)</td>
			<td style="width:71px;">Vector Labs</td>
			<td style="width:119px;">MP-7500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrogen peroxide</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">216763</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ImmPACT DAB substrate</td>
			<td style="width:71px;">Vector Labs</td>
			<td style="width:119px;">SK-4105</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hematoxylin counterstain</td>
			<td style="width:71px;">ThermoScientific</td>
			<td style="width:119px;">72404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Histochoice Mounting Media</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">H157-475</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Aperio Scanscope</td>
			<td style="width:71px;">Leica Biosystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Image Scanscope</td>
			<td style="width:71px;">Leica Biosystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Super HT PAP Pen</td>
			<td style="width:71px;">Research Products International</td>
			<td style="width:119px;">195506</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative immunohistochemistry of the cellular microenvironment in patient glioblastoma resections

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within patient samples of glioblastoma, the deadliest and most invasive brain cancer. Not only are quantitative methods beneficial for accurately describing diseased tissues, they can also potentially contribute to more accurate prognosis, diagnosis, and the development of tissue-engineered systems and replacements. In glioblastoma, glial cells, such as microglia and astrocytes, have been independently correlated with poor prognosis based on pathologist grading. However, the state of these cells and other glial cell components has not been well-described quantitatively. This can be difficult due to the large processes that mark these glial cells. Furthermore, most histological analyses focus on the overall tissue sample or only within the bulk of the tumor, as opposed to delineating quantifications based on regions within the highly heterogeneous tissue. Here, we describe a method for identifying and quantitatively analyzing the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations in patient tumor resections and ImageJ to analyze percent coverage of staining for each glial population. With these techniques we are able to better describe the glial cells throughout regions of the glioma tumor microenvironment.

For this analysis, two regions of interests within our tumor resections - the primary tumor bulk and the adjacent regions, primarily composed of healthy tissue with diffuse invading cancer cells (Figure 1A, 1B) - were identified by collaborating neuropathologists on hematoxylin and eosin stained patient samples. Within each patient sample, positive staining for astrocytes (Figure 1C), microglia (<strong class="xf...

Watch this Scientific Journal Video about Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections at JoVE.com

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

This protocol was developed to quantitatively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and ImageJ.

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

The overall goal of this protocol is to quantitively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and image J.This method can help answer key questions in the tumor microenvironment field, such as how these components look in patient samples and how we can determine physiologically relevant ratios of cells. The main advantage of this technique is that it is straightforward for analysis in patient samples. The implications of this technique extend toward elucidating the undercharacterized, complex glioblastoma tumor microenvironment.Generally, individuals new to this method will struggle because timing for chromogenic development needs to be closely monitored to prevent overdevelopment and saturation of the tissue. Begin the protocol by performing the washes specified in the accompanying text protocol. Dilute tris-based high pH antigen unmasking solution and distilled water using the manufacturers recommendations.Move on to heat mediated antigen retrieval by adding the diluted unmasking solution to a non-sealed microwaveable vessel. Place slides into the vessel. Then place the vessel slides into the microwave.Next, boil the vessel for 20 minutes at high power. Monitor the liquid levels for evaporation and replenish the vessel with distilled water as necessary. Allow samples to cool in solution for one hour at room temperature.Outline the tissue sample with a hydrophobic to minimize the volume of reagents necessary to cover the sample. Then, pipe in enough permeabilization solution, 100 to 200 microliters to cover the tissue sample. Remove and discard the solution and cover the tissue sample with permeabilization solution again.Incubate the samples at room temperature with blocking solution for one hour. Then, incubate the samples overnight in four degrees Celsius with primary antibodies diluted in blocking solution. The next day, detect primary antibodies using a horseradish peroxidase polymer reagent corresponding with the primary antibody host animal.Pipe in enough permeabilization solution to cover the tissue sample and incubate the sample for five minutes. Remove and discard solution and cover the tissue sample in permeabilization solution one more time for five minutes. Incubate the slides in 0.3%hydrogen peroxide and IX TBS for 15 minutes.Develop the samples with a peroxidase diaminobenzidine or dab substrate for two to 10 minutes until the desired stain intensity is achieved. Counterstain the samples to identify cell nuclei, such as with hematoxylin following the manufacturers protocol. Dehydrate samples with 100%ethanol and xylene.Finally, mount the samples permanently with mounting media. Two regions of interest within our tumor resections, the primary tumor Bulk and the Adjacent regions, primarily composed of healthy tissue with diffused invading cancer cells were identified by collaborating neuropathologist on hematoxylin and eosin stained patient samples. Chromogenic immunohistochemistry identified positive staining for astrocytes, microglia, and oligodendrocytes within each patient sample.We performed staining with no primary antibody for a negative control. The manufacturer recommended and further dilutions before settling on the one to 200 dilution due to optimal positive staining of cell body and process with minimizing background for the anti ALDH 1l1 antibody. Using Image J, the percent coverage of OSP-1 positive oligodendrocyte staining was quantified to eventually compare differences in our regions of interest.Once the appropriate threshold has been applied to the image, the percent coverage from the threshold can be measured and compared for multiple patients. Five adjacent and bulk regions of glioblastoma or GBM patient samples, along with healthy brain tissue were compared for oligodendrocyte-specific protein one or OSP1 percent coverage. Once mastered, the staining technique can be done in four hours on the first day and two hours the second day if it is performed properly.While attempting this procedure, it's important to remember to closely monitor the tissue staining intensity and hydration.

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9.5K vistas.  University of Virginia. The overall goal of this protocol is to quantitively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and image J.This method can help answer key questions in the tumor microenvironment field, such as how these components look in patient samples and how we can determine physiologically relevant ratios of cells. The main advantage of this technique is that it is straightforward for analysis in patient samples. The implications...