Name: elastic staining on paraffin-embedded slides of pt3n0m0 gastric cancer tissue
Uploaded: 2019-05-01T12:30:00
Description: Watch this Scientific Journal Video about Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue at JoVE.com

The authors thank the support in part from the National Natural Science Foundation (NO.81401902 and NO.81501992) and the Hunan Natural Science Foundation (NO.2017SK2134, NO.2018JJ2238). The manuscript is written by Guang Lei. The whole experiment is performed by Guang Lei and Haiyan Yang. The authors thank the assistance of pathologists and of fellows who follow up patients' data.

Between 1994 and 2005, patient samples were selected by two experienced gastrointestinal pathologists in the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, from patients who suffered gastrectomy of stomach neoplasms, with a postoperative pathological diagnosis of gastric cancer at the pT3N0M0 stage (according to the seventh TNM staging). This study was approved by the ethics review committee.
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	<li>Place the paraffin block into a fixed h...

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M., Casta&#241;o-Gonz&#225;lez, I., Mompe&#243;-Corredera, B., Ortega-Santana, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmentation+of+elastic+fibres+in+images+of+vessel+wall+sections+stained+with+Weigert's+resorcin-fuchsin.">Segmentation of elastic fibres in images of vessel wall sections stained with Weigert's resorcin-fuchsin.</a> Computer Methods and Programs in Biomedicine. 142, 43-54 (2017).</li><li>Kojima, M., Nakajima, K., Ishii, G., Saito, N., Ochiai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peritoneal+elastic+laminal+invasion+of+colorectal+cancer:+the+diagnostic+utility+and+clinicopathologic+relationship.">Peritoneal elastic laminal invasion of colorectal cancer: the diagnostic utility and clinicopathologic relationship.</a> American Journal of Surgical Pathology. 34 (9), 1351-1360 (2010).</li><li>Nakanishi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reappraisal+of+Serosal+Invasion+in+Patients+With+T3+Colorectal+Cancer+by+Elastic+Stain.">Reappraisal of Serosal Invasion in Patients With T3 Colorectal Cancer by Elastic Stain.</a> ARCHIVES OF PATHOLOGY &amp; LABORATORY MEDICINE. 140 (1), 81-85 (2016).</li><li>Grin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peritoneal+elastic+lamina+invasion:+limitations+in+its+use+as+a+prognostic+marker+in+stage+II+colorectal+cancer.">Peritoneal elastic lamina invasion: limitations in its use as a prognostic marker in stage II colorectal cancer.</a> Human Pathology. 44 (12), 2696-2705 (2013).</li><li>Stewart, C. 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This method proposed here provides an accurate approach for identifying the subserosal elastic lamina. The method can be used to evaluate whether tumor cells have invaded the elastic lamina in pT3N0M0 gastric cancer12. Elastic lamina is reddish in H&#38;E-stained sections, which could not be easily distinguished from collagen fibers, and other existing auxiliary diagnosis methods, such as cytology and immunohistochemistry, also exerted limited diagnostic value. The finding of tumor cells in the pe...

As per the Tumor, Node, and Metastasis (TNM) cancer staging system, the prognosis of gastric cancer at pathologic stage pT4 is significantly worse than that at pT3 (8th UICC/AJCC)1. With gastric cancer, classification of the primary tumor (pT) usually depends on the depth of tumor invasion2. Pathologic stage pT3 gastric cancer is defined as cancer invading through the muscularis propria into subserosal tissues, whereas pT4 gastric cancer is defined as cancer penetrating to the surface of the visceral peritoneum. The presence of the peritoneal surface invasiveness is the key to distinguish the two stages2. However, because the peritoneal mesothelial cell layer is very thin, it could be damaged during the surgery, postoperative treatment, and fixation3. Moreover, tumor-related inflammatory changes and fibrosis can damage the normal anatomy of the peritoneum, which makes it very difficult to accurately judge the peritoneal surface invasion using only H&#38;E staining4. Current auxiliary diagnosis methods such as cytology and immunohistochemistry are of limited diagnostic value5,6 because they are false-positive or have a low cost-effectiveness.
The serosal membrane includes the peritoneum, pleura, and pericardium, and is composed of the mesothelium, a basement membrane, and a sub-mesothelial layer7. One common histological feature of the serosal membrane is the presence of an elastic lamina in the sub-mesothelial layer, adjacent to the mesothelial cells8,9. Elastic lamina has a strong anti-damaging ability and can serve as an alternative prognostic marker if the mesothelial cells are destroyed by severe tumor-related inflammation or fibrosis3. Elastic lamina mainly comprises of elastin and microfibril10, which can be visualized by elastic staining. The principal reason behind the use of the elastic staining is that the elastin forms a hydrogen bond with the phenolic group of resorcinol in the elastin solution, causing elastic fibers to be stained blue-black. After Van Gieson (VG) contrasting staining, collagen fibers can be stained red and muscle fibers and red blood cells can be stained yellow8,11,12,13.
The eighth edition of the TNM staging of lung cancer defines visceral pleural invasion (T2) as a tumor invasion beyond the elastic lamina or an invasion at the surface of the visceral pleura2. Studies on colorectal cancer have shown that the elastic lamina invasion in pT3 colorectal cancer may be the reason for poor prognoses. The 5-year disease-free and overall survival rate of colorectal cancer have also been proven to be like pT4a13,14,15. Based on our previous studies on elastic staining, elastic lamina invasion has been found to have a significant negative influence on the prognosis of pT3 gastric cancer and should be treated the same way as pT4a gastric cancer12. Hence, this simple and cost-effective method can help eliminate uncertainty in results obtained through H&#38;E staining, and other common limitations associated with other auxiliary diagnosis methods, including cytology and immunohistochemistry, among others. This method can be used efficiently to diagnose peritoneal surface invasions of gastrointestinal cancer, especially in some ambiguous cases. It is convenient since H&#38;E stain and other stains usually make it difficult to identify such invasions. This method also ensures a reliability of results since it is very selective, particularly to elastic fibers. Here, we conduct elastic staining to determine whether tumor cells have invaded the elastic lamina in pT3N0M0 gastric cancer12.
This method is used for identifying elastic fibers in tissues on formalin-fixed, paraffin-embedded sections, and may be used for frozen sections as well. Here, we choose paraffin-embedded sections for the best maintenance of cellular and tissue morphology. All the steps in this protocol take place at room temperature. A commercial staining kit which includes a potassium permanganate solution, an oxalic acid solution, an elastin solution, and a Van Gieson&#39;s solution is used in the presented study.

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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:192px;">Elastin-Van Gieson staining kit</td>
			<td style="width:108px;">Baso</td>
			<td style="width:99px;">BA4083B</td>
			<td style="width:221px;">Used for staining of elastic fibers, collagen fibers and myofibers&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:192px;">Permount TM Mounting Medium</td>
			<td style="width:108px;">MXB Biotechnologies</td>
			<td style="width:99px;">DAB-0033</td>
			<td>Used for mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Ethanol</td>
			<td style="width:108px;">LMAI Bio&#160;</td>
			<td style="width:99px;">LM64-17-5&#160;</td>
			<td align="right">100%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Xylene</td>
			<td style="width:108px;">LMAI Bio&#160;</td>
			<td style="width:99px;">LX820585</td>
			<td></td>
		</tr>
	</tbody>
</table>

elastic staining on paraffin-embedded slides of pt3n0m0 gastric cancer tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

A successful elastic staining clearly reveals elastic lamina and cancer cells in pT3 gastric cancer. Figure 1 - low-power field, microscopically checked after elastin solution staining, prior to Van Gieson&#39;s counterstain as per the protocol mentioned - shows the elastic lamina is close to the serosal mesothelial cells and is stained blue-black in the form of filaments, and there is still a certain distance between the tumor cells and the elastic lamina. <...

Watch this Scientific Journal Video about Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue at JoVE.com

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

This method helps determine whether tumor cell invaded beyond elastic lamina in pT3 gastric cancer. elastic lamina erosion in the prognostic picture for pT3 gastric cancer. The main advantage of this technique is that it's convenient, and accurate approach to identify the elastic lamina, which makes it more precise for pathologist to evaluate whether tumor cells invade beyond elastic lamina in pT3 gastric cancer.This technique can be towards a pathologic test stage diagnosis of lung cancer and cancer. This method could also be applied to the TNM stage system of lung cancer and colo-rectal cancer. First, immerse the slides in xylene in a Coplin jar for 10 minutes.Remove the slides from this jar and immerse them in fresh xylene in a different Coplin jar for another 10 minutes. Immerse the slides in 100%ethanol for five minutes, and then transfer them to fresh 100%ethanol for another five minutes. Next, immerse the slides in 95%ethanol for two minutes.Transfer the slides to a jar containing 70%ethanol for two minutes. After this, rinse the slides briefly with distilled water in a new Coplin jar. First, use tissue paper to wipe off any excess solution on the slide or around the tissue.Place the slides in conditions that match room temperature and humidity. Add a few drops of potassium permanganate to each slide to oxidize them, making sure that the volume added is enough to cover the tissue section on each slide. After five minutes, rinse the oxidized slides with distilled water in Coplin jars, repeating the rinse two to three times and using a new jar of water for each rinse.The tissue should have an observable brown color. Now, add a few drops of oxalic acid to each slide to bleach them, making sure that the volume added is enough to cover the tissue section on each slide. After five minute, rinse the bleached slides with distilled water, repeating the rinse two to three times and using a new jar of water for each rinse.Wash the slides briefly in 95%alcohol. Immerse the washed slides in elastin solution for eight to 24 hours. Then immerse the slides directly in 95%ethanol for one to two minutes to differentiate each tissue section well.Rinse the slides completely with distilled water. Check microscopically for the blue-black elastic fibers staining and the gray background. Counterstain the slides in a van Gieson solution for one minute, making sure that the volume used is enough to cover the tissue section on each slide completely.Next, rapidly drop 95%ethanol onto the tissue slices for a few seconds to differentiate each tissue section well. To begin, immerse the slides in 95%alcohol for five minutes to dehydrate them quickly. Transfer the slides to 100%alcohol for five minutes to continue the dehydration.Then, transfer the slides to fresh 100%alcohol for five additional minutes. Immerse the slides in two different changes of xylene with each immersion lasting for three minutes. After this, add one drop of resinous mounting medium to the slide and place a cover slip on top.Using a microscope, observe the tissue sections as outlined in the text protocol. In this study, elastic staining is utilized to visually identify subserosal elastic lamina. A representative successful staining reveals that after elastin solution staining, but before van Gieson's counterstaining, the elastic lamina can be clearly seen in the form of filaments that are stained blue-black.They are close to the serosal mesothelial cells, and a certain amount of distance is still seen between the elastic lamina and the tumor cells. Typical elastic stainings with van Gieson's counterstain demonstrate that the elastic lamina is stained blue-black. The collagen fibers are stained red, and the muscle fibers and cancer cells are stained yellow.Cancer cells penetrating the elastic lamina is considered elastic lamina invasion. Whereas cancer cells being close to but not invading the elastic lamina is considered elastic lamina non-invasion. If the staining reveals a repetitive structure indicative of multiple layers of elastic lamina, and the tumor does not invade beyond the outermost layer, the sample should be diagnosed as elastic lamina non-invasion.Elastin solution is a and the staining time is relative long, so it's best to immerse all slides with the elastin staining in a sealed container. After elastin solution staining, should be washed away with distilled water.

elastic-staining-on-paraffin-embedded-slides-pt3n0m0-gastric-cancer

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Deciphering Tumor Microenvironment Using mIHC in Lung Squamous Carcinoma

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been considered the leading cause of death in lung cancer patients. The complexity of the tumor microenvironment requires effective methods to understand cell-to-cell relationships in tumor tissues. The multiplex immunohistochemistry (mIHC) technique has become a key tool for inferring the relationship between the expression of proteins upstream and downstream of signaling pathways in tumor tissues and developing clinical diagnoses and treatment plans. mIHC is a multi-label immunofluorescence staining method based on Tyramine Signal Amplification (TSA) technology, which can simultaneously detect multiple target molecules on the same tissue section sample to achieve different protein co-expression and co-localization analysis. In this experimental protocol, paraffin-embedded tissue sections of lung squamous carcinoma of clinical origin were subjected to multiplex immunohistochemical staining. By optimizing the experimental protocol, multiplex immunohistochemical staining of labeled target cells and proteins was achieved, solving the problem of autofluorescence and channel crosstalk in lung tissues. In addition, multiplex immunohistochemical staining is widely used in the experimental validation of tumor-related, high-throughput sequencing, including single-cell sequencing, proteomics, and tissue space sequencing, providing intuitive and visual pathology validation results.

Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample

This experimental protocol describes and optimizes a multiplex immunohistochemistry (IHC) staining method, mainly by optimizing single-channel antibody incubation conditions and adjusting the settings of antibodies and channels to solve the problems of autofluorescence and channel crosstalk in lung cancer tissues of clinical origin.

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been ...

In Situ Immunohistochemical Detection of Immune Cell Markers in Cancer Tissues

Multiplex Cyclic Fluorescent Immunohistochemistry

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry

Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and ...

FACS Analysis of Lipid Hydroperoxides in Ferroptotic Medulloblastoma Cells

Revealing the Ferroptotic Phenotype of Medulloblastoma

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and puzzle, leading to new biological ventures. In 2012, a Columbia University group recognized this interaction as a central event leading to a new type of regulated cell death named &#34;ferroptosis.&#34; The major feature of ferroptosis is the accumulation of lipid hydroperoxides due to (1) dysfunctional antioxidant defense and/or (2) overwhelming oxidative stress, which most frequently coincides with increased content of free labile iron in the cell. This is normally prevented by the canonical anti-ferroptotic axis comprising the cystine transporter xCT, glutathione (GSH), and GSH peroxidase 4 (GPx4). Since ferroptosis is not a programmed type of cell death, it does not involve signaling pathways characteristic of apoptosis. The most common way to prove this type of cell death is by using lipophilic antioxidants (vitamin E, ferrostatin-1, etc.) to prevent it. These molecules can approach and detoxify oxidative damage in the plasma membrane. Another important aspect in revealing the ferroptotic phenotype is detecting the preceding accumulation of lipid hydroperoxides, for which the specific dye BODIPY C11 is used. The present manuscript will show how ferroptosis can be induced in wild-type medulloblastoma cells by using different inducers: erastin, RSL3, and iron-donor. Similarly, the xCT-KO cells that grow in the presence of NAC, and which undergo ferroptosis once NAC is removed, will be used. The characteristic &#34;bubbling&#34; phenotype is visible under the light microscope within 12-16 h from the moment of ferroptosis triggering. Furthermore, BODIPY C11 staining followed by FACS analysis to show the accumulation of lipid hydroperoxides and consequent cell death using the PI staining method will be used. To prove the ferroptotic nature of cell death, ferrostatin-1 will be used as a specific ferroptosis-preventing agent.

Author Spotlight: Tracing the Ferroptotic Signatures and Cell Death Dynamics in Medulloblastoma for Advanced Therapeutics 

Lipid hydroperoxide content represents the most commonly used indicator of ferroptotic cell death. This article demonstrates the step-by-step flow cytometry analysis of lipid hydroperoxide content in cells upon ferroptosis induction.

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and ...

Optimizing Multiplex Immunohistochemistry (mIHC) Staining for Lung Cancer Tissue Samples to Address Autofluorescence Issues

To begin, obtain previously prepared formula and fixed and paraffin embedded lung cancer tissue sample slide. Incubate it at 65 degrees Celsius for five minutes. Perform dehydration by immersing the slide in xylene, followed by ethanol solutions of decreasing concentrations.Then immerse the slides in sterilized water three times for one minute each. For antigen retrieval, place the slide in a staining and repair box. Fill it with immunohistochemical antigen repair buffer working solution of pH six or nine.Cover the box with a lid, and heat it in a microwave oven for eight minutes at 100%power. Then cool the slide to room temperature. Cover the tissue with a blocking solution, and then incubate in a humidified chamber at room temperature for 10 minutes.After removing the blocking solution, cover the slide with the primary antibody working solution, and place it in a humidified chamber. Then incubate this chamber for one hour at 37 degrees Celsius. Next, wash the slide with wash buffer working solution for two minutes.Add polymer horseradish peroxidase directly dropwise onto the slide, and incubate it for 15 minutes at room temperature. After washing the slide, add fluorophore working solution directly dropwise onto the slide, and incubate it for 10 minutes at room temperature. Once the slide is washed, give microwave treatment as demonstrated previously.When the slide is cooled to room temperature, wash it three times with double distilled water for one minute each. Next, dip the slide in the wash buffer working solution for two minutes, and perform antibody incubation, horseradish peroxidase, and fluorophore addition as demonstrated. For cell nuclear staining, cover the slide with DAPI working solution and incubate it in a humidified chamber for five minutes at room temperature.Then wash the slides with double distilled water three times, one minute each. Remove water droplets from the slide. Add 10 to 20 microliters of anti-fluorescence quencher to the slide, and seal it with a microscope cover glass.