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.
1. Sectioning
<ol>
	<li>Place the paraffin block into a fixed holder of the sledge microtome, which can move backward and forward across the blade. Adjust the optimal angle between the block and the microtome knife. 
	NOTE: The optimal angle not only depends on the blade&#39;s geometry but also on the cutting speed and technique.</li>
	<li>Trim the paraffin block as necessary and cut it into 5-&#181;m-thick cross sections.</li>
	<li>Place the paraffin 5-&#181;m sections in a water bath at about 40 - 45 &#176;C and mount it, subsequently, from the water onto the glass slide, one section per slide.</li>
	<li>Press the mounted section carefully against a paper towel to remove any residual water and potential air bubbles.</li>
	<li>Allow sections to air-dry for 30 min and then bake them in an oven at 45 - 50 &#176;C for 2 h.</li>
</ol>
2. Deparaffinization and Rehydration of All Slides
<ol>
	<li>Immerse the slides in xylene in a Coplin jar for 10 min.</li>
	<li>Take the slides out from the first xylene jar and immerse them in xylene in another jar for 10 min.</li>
	<li>Now, immerse the slides in 100% ethanol in a Coplin jar for 5 min.</li>
	<li>Immerse the slides in 100% ethanol in another Coplin jar, also for 5 min.</li>
	<li>Immerse the slides in 95% ethanol in a Coplin jar for 2 min.</li>
	<li>Immerse the slides in 70% ethanol in a Coplin jar for 2 min. 
	NOTE: Ensure that the slides are completely immersed in the solutions while performing the above steps.</li>
	<li>Rinse the slides briefly with distilled water in a new jar.</li>
</ol>
3. Staining
<ol>
	<li>Before proceeding for staining, wipe off any excess solutions around the tissue on the slide with a tissue paper.</li>
	<li>Place the slides inroom temperature and room humidity conditions to avoid drying of the tissue on the slide throughout the entire staining process.</li>
	<li>Oxidize the slides with a few drops of potassium permanganate for 5 min. Ensure that the volume of potassium permanganate is enough to cover the tissue section on each slide.</li>
	<li>Rinse the oxidized slides with distilled water in a Coplin jar with 2 - 3 changes. Observe the blue color of the tissue.</li>
	<li>Now, bleach these slides by adding a few drops of oxalic acid for 5 min. Ensure that the volume of oxalic acid is enough to completely cover the tissue section on each slide.</li>
	<li>Rinse the slides by immersing them in the Coplin jar filled with distilled water. Perform the rinse 2x - 3x.</li>
	<li>Now, wash these slides briefly in 95% alcohol, and then immerse the slides into an elastin solution (5 g/L) for 8 - 24 h. 
	NOTE: Elastin solution is volatile, and its staining time is relatively long, so it is best to immerse the slides with elastic staining in a sealed container, such as a transparent glass bottle with a sealing cap. The capacity of the container and the volume of the elastin solution depends on the number of immersed slides, as long as it enables the complete immersion of all the slides.</li>
	<li>Immerse these slides directly in 95% ethanol for 1 - 2 min to differentiate each tissue section well. 
	NOTE: Differentiation refers to the change in the charge of the tissue section, which removes the excess staining absorbed by the tissue and makes the colors clearer. After elastin solution staining, samples should not be washed with distilled water to avoid any difficulty in differentiation. Check microscopically, as necessary, for blue-black elastic fibers staining and gray background. It is better to slightly underdifferentiate the tissue since the subsequent Van Gieson&#39;s counterstain can also somewhat extract the elastic stain.</li>
	<li>Fully rinse these slides with distilled water after differentiation.</li>
	<li>Counterstain the slides in a Van Gieson&#39;s solution for 1 min. 
	NOTE: The volume of the Van Gieson&#39;s solution needs to be enough to be able to immerse the tissue section on each slide completely.</li>
	<li>Rapidly drop 95% ethanol onto these slices for a few seconds to differentiate each tissue section well. 
	NOTE: Do not rinse the slides with any water after the Van Gieson&#39;s counterstaining, as that could weaken or even elute the color of the Van Gieson&#39;s counterstaining.</li>
</ol>
4. Dehydration and Mounting
<ol>
	<li>Immerse the slides in 95% alcohol for 5 min to let them dehydrate quickly.</li>
	<li>Continue to dehydrate these slices by 2 changes of 100% alcohol, each time immersing the slides for 5 min.</li>
	<li>Immerse the slides into 2 changes of xylene, each time for 3 min.</li>
	<li>Add one drop of resinous mounting medium to the slide and place the coverslip on the top.</li>
</ol>
5. Microscopic Observation of the Slides
<ol>
	<li>Find the serosal direction and observe the results of elastic staining: elastic fibers (lamina) are stained blue-black, collagen fibers are stained red, and muscle fibers are stained yellow.</li>
	<li>Look for the cancer cells in the whole slide: cancer cells are stained yellow12.</li>
	<li>Determine the positional relationship between the cancer cells and elastic lamina. 
	NOTE: There will be two situations here. As long as there is one tumor cell that has invaded beyond the elastic lamina in the whole slide, then this case could be considered as elastic lamina invasion. On the other hand, only when there is no tumor cell that has invaded beyond the elastic lamina in the whole slide, this case could be considered as elastic lamina non-invasion.</li>
</ol>

<ol><li>Jiang, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinicopathologic+characteristics+and+prognosis+of+gastric+cancer+invading+the+subserosa%5BTitle%5D)+AND+%22Journal+of+Surgical+Oncology%22%5BJournal%5D)">Clinicopathologic characteristics and prognosis of gastric cancer invading the subserosa.</a> Journal of Surgical Oncology. 102 (7), 737-741 (2010).</li>
<li>Amin, M. B., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAmin%2C+MB+%22AJCC+Cancer+Staging+Manual.+8th+ed%22">AJCC Cancer Staging Manual. 8th ed</a>. , Springer. New York, NY. (2017).</li>
<li>Kojima, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elastic+laminal+invasion+in+colon+cancer%3A+diagnostic+utility+and+histological+features%5BTitle%5D)+AND+%22Frontiers+in+Oncology%22%5BJournal%5D)">Elastic laminal invasion in colon cancer: diagnostic utility and histological features.</a> Frontiers in Oncology. 2, 179(2012).</li>
<li>Ludeman, L., Shepherd, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serosal+involvement7+in+gastrointestinal+cancer%3A+its+assessment+and+significance%5BTitle%5D)+AND+%22Histopathology%22%5BJournal%5D)">Serosal involvement7 in gastrointestinal cancer: its assessment and significance.</a> Histopathology. 47 (2), 123-131 (2005).</li>
<li>Shepherd, N. A., Baxter, K. J., Love, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+prognostic+importance+of+peritoneal+involvement+in+colonic+cancer%3A+a+prospective+evaluation%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">The prognostic importance of peritoneal involvement in colonic cancer: a prospective evaluation.</a> Gastroenterology. 112 (4), 1096-1102 (1997).</li>
<li>Liang, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retrospective+evaluation+of+elastic+stain+in+the+assessment+of+serosal+invasion+of+pT3N0+colorectal+cancers%5BTitle%5D)+AND+%22American+Journal+of+Surgical+Pathology%22%5BJournal%5D)">Retrospective evaluation of elastic stain in the assessment of serosal invasion of pT3N0 colorectal cancers.</a> American Journal of Surgical Pathology. 37 (10), 1565-1570 (2013).</li>
<li>Carter, D., True, L., Otis, C. N. Serosal membranes. <a target="_blank" href="https://www.amazon.com/Histology-Pathologists-Stacey-Mills-MD/dp/0781762413">Histology for Pathologists. 3rd ed</a>. Mills, S. E. , Lippincott Williams and Wilkins. Philadelphia, PA. 547-562 (2007).</li>
<li>Knudsen, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+peritoneal+elastic+lamina%5BTitle%5D)+AND+%22Journal+of+Anatomy%22%5BJournal%5D)">The peritoneal elastic lamina.</a> Journal of Anatomy. 177, 41-46 (1991).</li>
<li>Shepherd, N. A., Baxter, K. J., Love, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+local+peritoneal+involvement+on+pelvic+recurrence+and+prognosis+in+rectal+cancer%5BTitle%5D)+AND+%22Journal+of+Clinical+Pathology%22%5BJournal%5D)">Influence of local peritoneal involvement on pelvic recurrence and prognosis in rectal cancer.</a> Journal of Clinical Pathology. 48 (9), 849-855 (1995).</li>
<li>Uitto, J., Li, Q., Urban, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+complexity+of+elastic+fibre+biogenesis+in+the+skin--a+perspective+to+the+clinical+heterogeneity+of+cutis+laxa%5BTitle%5D)+AND+%22Experimental+Dermatology%22%5BJournal%5D)">The complexity of elastic fibre biogenesis in the skin--a perspective to the clinical heterogeneity of cutis laxa.</a> Experimental Dermatology. 22 (2), 88-92 (2013).</li>
<li>Kazlouskaya, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+utility+of+elastic+Verhoeff-Van+Gieson+staining+in+dermatopathology%5BTitle%5D)+AND+%22Journal+of+Cutaneous+Pathology%22%5BJournal%5D)">The utility of elastic Verhoeff-Van Gieson staining in dermatopathology.</a> Journal of Cutaneous Pathology. 40 (2), 211-225 (2013).</li>
<li>Lei, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elastic+staining-a+rejuvenated+method+to+reassess+prognosis+and+serosal+invasion+in+patients+with+pT3N0M0+gastric+cancer%5BTitle%5D)+AND+%22Human+Pathology%22%5BJournal%5D)">Elastic staining-a rejuvenated method to reassess prognosis and serosal invasion in patients with pT3N0M0 gastric cancer.</a> Human Pathology. 65, 79-84 (2017).</li>
<li>Hernández-Morera, P., Travieso-González, C. M., Castaño-González, I., Mompeó-Corredera, B., Ortega-Santana, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Segmentation+of+elastic+fibres+in+images+of+vessel+wall+sections+stained+with+Weigert%27s+resorcin-fuchsin%5BTitle%5D)+AND+%22Computer+Methods+and+Programs+in+Biomedicine%22%5BJournal%5D)">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/pubmed?term=((Peritoneal+elastic+laminal+invasion+of+colorectal+cancer%3A+the+diagnostic+utility+and+clinicopathologic+relationship%5BTitle%5D)+AND+%22American+Journal+of+Surgical+Pathology%22%5BJournal%5D)">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/pubmed?term=((Reappraisal+of+Serosal+Invasion+in+Patients+With+T3+Colorectal+Cancer+by+Elastic+Stain%5BTitle%5D)+AND+%22ARCHIVES+OF+PATHOLOGY+%26+LABORATORY+MEDICINE%22%5BJournal%5D)">Reappraisal of Serosal Invasion in Patients With T3 Colorectal Cancer by Elastic Stain.</a> ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE. 140 (1), 81-85 (2016).</li>
<li>Grin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peritoneal+elastic+lamina+invasion%3A+limitations+in+its+use+as+a+prognostic+marker+in+stage+II+colorectal+cancer%5BTitle%5D)+AND+%22Human+Pathology%22%5BJournal%5D)">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. J., Brennan, B. A., Crook, M. L., Russell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Value+of+elastin+staining+in+the+assessment+of+peritoneal+implants+associated+with+ovarian+serous+borderline+tumours%5BTitle%5D)+AND+%22Histopathology%22%5BJournal%5D)">Value of elastin staining in the assessment of peritoneal implants associated with ovarian serous borderline tumours.</a> Histopathology. 51 (3), 313-321 (2007).</li>
<li>Kojima, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathological+diagnostic+criterion+of+blood+and+lymphatic+vessel+invasion+in+colorectal+cancer%3A+a+framework+for+developing+an+objective+pathological+diagnostic+system+using+the+Delphi+method%2C+from+the+Pathology+Working+Group+of+the+Japanese+Society+for+Cancer+of+the+Colon+and+Rectum%5BTitle%5D)+AND+%22Journal+of+Clinical+Pathology%22%5BJournal%5D)">Pathological diagnostic criterion of blood and lymphatic vessel invasion in colorectal cancer: a framework for developing an objective pathological diagnostic system using the Delphi method, from the Pathology Working Group of the Japanese Society for Cancer of the Colon and Rectum.</a> Journal of Clinical Pathology. 66 (7), 551-558 (2013).</li>
<li>Elston, C. A., Kazlouskaya, V., Elston, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elastic+staining+versus.+fluorescent+and+polarized+microscopy+in+the+diagnosis+of+alopecia%5BTitle%5D)+AND+%22Journal+of+the+American+Academy+of+Dermatology%22%5BJournal%5D)">Elastic staining versus. fluorescent and polarized microscopy in the diagnosis of alopecia.</a> Journal of the American Academy of Dermatology. 69 (2), 288-293 (2013).</li>
<li>Vos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predominance+of+Nonatherosclerotic+Internal+Elastic+Lamina+Calcification+in+the+Intracranial+Internal+Carotid+Artery%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Predominance of Nonatherosclerotic Internal Elastic Lamina Calcification in the Intracranial Internal Carotid Artery.</a> Stroke. 47 (1), 221-223 (2016).</li>
</ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>Elastin-Van Gieson staining kit</td><td>Baso</td><td>BA4083B</td><td>Used for staining of elastic fibers, collagen fibers and myofibers&amp;nbsp;</td></tr><tr><td>Permount TM Mounting Medium</td><td>MXB Biotechnologies</td><td>DAB-0033</td><td>Used for mounting</td></tr><tr><td>Ethanol</td><td>LMAI Bio&amp;nbsp;</td><td>LM64-17-5&amp;nbsp;</td><td>100%</td></tr><tr><td>Xylene</td><td>LMAI Bio&amp;nbsp;</td><td>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 ...

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7.0K Views.  Central South University. Deze methode helpt bepalen of tumorcel binnengedrongen dan elastische lamina in pT3 maagkanker. elastische lamina erosie in het prognostische beeld voor pT3 maagkanker. Het belangrijkste voordeel van deze techniek is dat het handig is, en nauwkeurige aanpak om de elastische lamina te identificeren, waardoor het nauwkeuriger voor patholoog om te evalueren of tumorcellen binnenvallen dan elastische lamina in pT3 maagkanker.Deze techniek kan worden in de richting van een pathologische tes...

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

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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 ...

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.
Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.
DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.
We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

Biology

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Medicine

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Neuroscience

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...

Therapeutic Effects of Huazhuojiedu Decoction on Precancerous Lesions of Gastric Cancer by Regulating Mitophagy

This research aims to explore the therapeutic effect and potential mechanisms of Huazhuojiedu decoction (HZJD) for alleviating precancerous lesions of gastric cancer (PLGC) both in vivo and in vitro. HZJD is a traditional Chinese herbal formula consisting of 11 herbs. Sprague-Dawley (SD) rats were randomly divided into four subgroups: control group, model group, positive drug group, and HZJD group. Hematoxylin-eosin (H&amp;E) staining, high iron diamine-alcian blue (HID-AB) staining, alcian blue-periodic acid Schiff (AB-PAS) staining, immunohistochemistry, immunofluorescence, RT-qPCR, and Western blot assays were performed after 10 weeks of HZJD treatment. In vitro, the cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to detect cell proliferation. RT-qPCR and Western blot assays were performed to evaluate mitophagy levels. The results indicated that HZJD could retard the pathological progression in PLGC rats and reduce PLGC cell proliferation. Treatment with HZJD significantly increased the mRNA and protein expression levels of Sirt3, Foxo3a, Parkin, and LC3 II/I, while decreasing the mRNA and protein expression levels of p62 and Tomm20. HZJD was found to have the ability to reverse the decline in mitophagy activity both in vivo and in vitro. In conclusion, the study assessed the impact of HZJD and provided evidence regarding its potential molecular mechanism.

Here, we introduce a protocol that demonstrates the therapeutic efficacy of Huazhuojiedu decoction (HZJD) in alleviating precancerous lesions of gastric cancer using mitophagy regulation.

This research aims to explore the therapeutic effect and potential mechanisms of Huazhuojiedu decoction (HZJD) for alleviating precancerous lesions of ...

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

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