The authors have nothing to disclose.

All procedures including human subjects have been approved by Dharmais Cancer Hospital Ethics Committee with certificate number of 040/KEPK/VII/2017. All patients signed the consent forms and expressed agreement to participate in this study.
NOTE: The inclusion criteria are patients with diagnosis of early breast cancer, with tumor stage T1-T2 without palpable and ultrasonography lymph nodes enlargement (cNo). The exclusion criteria are locally advanced breast cancer, received neo adjuvant chemo or hormonal therapy, and pregnancy.
1. Preparation of 1% methylene blue dye and the injection technique
<ol>
	<li>Sterilize the surgical field after anesthetizing the patient.</li>
	<li>Aspirate 2 mL of 1% methylene blue dye from its vial with a 3 mL syringe.</li>
	<li>Draw a line to mark the lower axillary hairline below the lateral border of pectoralis major muscle.</li>
	<li>Inject 2 mL of 1% methylene blue dye into the subareolar space of the breast.</li>
	<li>If there is a scar in the upper outer quadrant of the breast or NAC, inject 2 mL of 1% methylene blue dye peritumorally into the breast parenchyma at the lateral side of the scar towards the axilla with a 23G needle under ultrasound guidance with a linear probe (12 MHz).</li>
	<li>Massage the breast circularly at the injection site for 5 min, and then continue to perform surgery.</li>
</ol>
2. Sentinel node biopsy technique in breast conserving surgery (BCS)
NOTE: The surgery is performed in a patient who underwent BCS and SNB.
<ol>
	<li>Prepare the surgical tools: monopolar electrocautery, DeBakey forceps/anatomical forceps, and retractors.</li>
	<li>Incise the skin, subcutaneous tissue, and fascia.</li>
	<li>Find the blue nodes or blue lymphatic tracts. Follow the blue tracts until the blue nodes or non-blue nodes with lymphatic blue tracts are identifiable.</li>
	<li>Search for the sentinel nodes along the intercostobrachial nerve and lateral thoracic vein if the blue nodes or blue lymphatic tracts cannot be found.</li>
	<li>Resect the sentinel nodes carefully and avoid damaging the nodes.</li>
	<li>Palpate the axillary space to find additional suspicious malignant lymph node enlargement.</li>
</ol>
3. Sentinel node biopsy technique in mastectomy
NOTE: The surgery is done in a patient who underwent mastectomy and SNB.
<ol>
	<li>Incise the skin and subcutaneous tissue.</li>
	<li>Create skin flaps.</li>
	<li>Remove the breast from pectoralis major until axillary fossa can be fully exposed.</li>
	<li>Incise the clavicopectoral fascia to find the sentinel node.</li>
	<li>If the blue lymphatic tracts cannot be found, find the sentinel node along the intercostobrachial nerve and lateral thoracic vein area.</li>
	<li>Remove the sentinel node.</li>
	<li>Look for additional suspicious lymph nodes by palpation.</li>
</ol>
4. Intraoperative examination
<ol>
	<li>Slice the lymph nodes no thicker than 2 mm, parallel to the long axis.</li>
	<li>Make touch imprint cytology from each node.</li>
	<li>Place the surgical specimen on a metal tissue disc and embed in a gel-like medium with the same density as frozen tissue.</li>
	<li>Submit all of the nodes for frozen section (FS) examination.</li>
	<li>Categorize the metastatic status of sentinel nodes into positive or negative, and report it to the surgeon during the surgery.</li>
</ol>
5. Pathological examination
<ol>
	<li>Perform the final pathologic evaluation of the sentinel nodes on formalin-fixed and paraffin-embedded tissue sections.</li>
	<li>Classify the sentinel nodes metastasis according to the 6th edition of American Joint Committee on Cancer (AJCC) manual. Macrometastasis (MAC) is defined as tumor deposits larger than 2 mm, micrometastasis (MiC) is defined as tumor deposits between 0.2 and 2 mm, isolated tumor cells (ITC) are defined as cell clusters no larger than 0.2 mm.</li>
	<li>Perform the serial sectioning and immunohistochemistry analysis for cytokeratin when there are doubts over defining ITC.</li>
	<li>Examine the rest of axillary lymph nodes in a similar manner.</li>
</ol>

<ol>
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J., Ramadhan, ., 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=Methylene+blue+dye+as+a+single+agent+in+breast+cancer+sentinel+lymph+node+biopsy:+initial+study+of+cancer+centre+hospital.">Methylene blue dye as a single agent in breast cancer sentinel lymph node biopsy: initial study of cancer centre hospital.</a> 19th Asian Congress of Surgery &amp; SingHealth Surgical Congress. , (2013).</li><li>Borgstein, P. J., Meijer, S., Pijpers, R. J., van Diest, P. 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We thank Muhammad, M.D. for preparing and assisting the surgery, Mr. Ali Abdul Aziz, Mr. Adhitya Bayu, and Mr. Ariananda Hariadi in helping with the manuscript preparation. We also acknowledge Mr. Tegar Kharisma for his help on video editing.

In the modern era of breast cancer surgery, SNB has replaced ALND as the standard of care for axillary staging in early breast cancer and ALND should be abandoned if the SNs are free from metastasis14,15. The lymphatic mapping technique which is commonly used in developed countries is the application of radioisotope tracer and PBD as a combined or single technique16,17. The question on how to perform SNB ...

The status of axillary lymph nodes (ALNs) metastasis is the most important prognostic factor in breast cancer. Axillary lymph node dissection (ALND) was the conventional procedure to assess metastatic status of ALNs1,2. Unfortunately, ALND results in morbidities to the patients which decreases the quality of life, especially by increasing the risk of lymphedema after this procedure3,4. Nowadays, sentinel node biopsy (SNB) has replaced ALND for axillary staging because of its minimal morbidities among patients5. The most common method for performing SNB is using radioisotope tracer and PBD6. In some parts of the world, including in developing countries, these tracer agents could be difficult to procure and searching for an alternative tracer agent is critical to solve the problem.
Initially, MBD was used by Wong et al. as a tracer agent for mapping sentinel nodes (SNs)7. In their study using a feline model, intradermal injection of MBD showed poor lymphatic uptake and isosulfan blue was chosen as the preferred dye for sentinel node (SN) mapping7. Methylene blue dye has been used in breast cancer SNB since the first successful report by Simmons et al.8. Several studies have also reported MBD as the favorable dye for SNs identification, and that the false negative rates of MBD technique were comparable to radioisotope or PBD9,10,11. Fewer allergic reactions and lower price are the other reasons to consider its use in SN mapping12.
Recently, we studied the use of 1% MBD alone for SNB in clinically node negative breast cancer. In early stages, MBD has a favorable identification rate and negative predictive value13. We inject 2 mL of 1% MBD into the subareolar space or peritumoral area if there was an excisional biopsy scar at the upper outer quadrant or nipple areolar complex (NAC) of the breast. The blue nodes and non-blue nodes with lymphatic blue channels are categorized as SNs. The anatomical landmark in the axilla is used as a guidance to find SNs. Intraoperative examination is applied to assess the metastasis and the SNs are sent for histopathology analysis based on American Society of Clinical Oncology (ASCO) Guidelines14.
If the cases are selected carefully and the skills required for this technique are obtained by the surgeons as well as pathologists, many patients could be saved from the harmful effects of ALND while still having favorable survival.

<table><tbody><tr><td>Metiblo 50mg/5ml</td><td>Laboratories Sterop NV</td><td>BE475217</td><td>Methylene Blue Dye</td></tr><tr><td>Disposable syringe with needle - 3mL (Luer Lock Tip)</td><td>Terumo Europe NV</td><td>dvr-3414</td><td>Syringe for Injection</td></tr><tr><td>ForceTriad energy platform</td><td>Medtronic</td><td>ForceTriad</td><td>Surgical cautery</td></tr><tr><td>Shandon Cryomatrix embedding resin</td><td>Thermo (scientific)</td><td>6769006</td><td>Frozen Section</td></tr><tr><td>Cryotome FSE</td><td>Thermo (scientific)</td><td>77210153</td><td></td></tr><tr><td>HistoStar Embedding Workstation</td><td>Thermo (scientific)</td><td>A81000001</td><td>Histopathological Examination</td></tr><tr><td>Finesse Me+</td><td>Thermo (scientific)</td><td>A77510272</td><td></td></tr><tr><td>Gemini AS</td><td>Thermo (scientific)</td><td>A81500002</td><td></td></tr><tr><td>Benchmark GX</td><td>Ventana Medical Systems</td><td>750-850</td><td>Immunohistochemistry</td></tr><tr><td>Benchmark XT</td><td>Ventana Medical Systems</td><td>750-700</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>Model BX53</td><td></td></tr><tr><td>Ultrasound</td><td>Phillips</td><td>HD 7XE</td><td></td></tr></tbody></table>

the application of 1% methylene blue dye as a single technique in breast cancer sentinel node biopsy

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery (BCS), a separate incision in the lower axilla hairline was made to find the sentinel nodes (SNs). In mastectomy, the SNs were identified through the same mastectomy incision. The SNs were described as blue nodes or nodes with lymphatic blue channels. An anatomical landmark in the axilla was used to facilitate SNs identification. The SNs metastases were evaluated by intraoperative frozen section analysis and histopathology examination as it is a gold standard. Here, we described the MBD as the lone technique in breast cancer sentinel node biopsy (SNB) which could be useful when radioisotope tracer or patent or isosulfan blue dye (PBD) cannot be provided.

Here, we describe the results from the presented technique. Two milliliters of 1% MBD were injected at the deep subareolar space as the standard technique of injection (Figure 1A). If the peritumoral injection is indicated, it should be performed under ultrasound guidance (Figure 1B). The blue nodes or lymphatic blue tracts were seen after entering the axillary space. Following the lymphatic blue tracts lead to finding the SNs (<...

Watch this Scientific Journal Video about The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy at JoVE.com

The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs).

In this study, we injected 1% methylene blue dye (MBD) into the subareolar or peritumoral space of the breast. In the case of breast conserving surgery ...

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18.9K Views.  Dharmais Cancer Hospital. In Indonesia, sentinel node biopsy (SNB) is not routinely performed for breast cancer surgery because of the limitation to provide radioisotope tracer and isosulfan or patent blue dye (PBD). To overcome this obstacle, we applied 1% methylene blue dye (MBD) as a single agent to map the sentinel nodes (SNs). 

Video: The Application of 1% Methylene Blue Dye As a Single Technique in Breast Cancer Sentinel Node Biopsy

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Medicine

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Intraoperative Detection of Subtle Endometriosis: A Novel Paradigm for Detection and Treatment of Pelvic Pain Associated with the Loss of Peritoneal Integrity

Endometriosis is a common disease affecting 40 to 70% of reproductive-aged women with chronic pelvic pain (CPP) and/or infertility. The purpose of this study was to demonstrate the use of a blue dye (methylene blue) to stain peritoneal surfaces during laparoscopy (L/S) to detect the loss of peritoneal integrity in patients with pelvic pain and suspected endometriosis. Forty women with CPP and 5 women without pain were evaluated in this pilot study. During L/S, concentrated dye was sprayed onto peritoneal surfaces, then aspirated and rinsed with Lactated Ringers solution. Areas of localized dye uptake were evaluated for the presence of visible endometriotic lesions. Areas of intense peritoneal staining were resected and some fixed in 2.5% buffered gluteraldehyde and examined by scanning (SEM) electron microscopy. Blue dye uptake was more common in women with endometriosis and chronic pelvic pain than controls (85% vs. 40%). Resection of the blue stained areas revealed endometriosis by SEM and loss of peritoneal cell-cell contact compared to normal, non-staining peritoneum. Affected peritoneum was associated with visible endometriotic implants in most but not all patients. Subjective pain relief was reported in 80% of subjects. Based on scanning electron microscopy, we conclude that endometrial cells extend well beyond visible implants of endometriosis and appear to disrupt the underlying mesothelium. Subtle lesions of endometriosis could therefore cause pelvic pain by disruption of peritoneal integrity, allowing menstrual or ovulatory blood and associated pain factors access to underlying sensory nerves. Complete resection of affected peritoneum may provide a better long-term treatment for endometriosis and CPP. This simple technique appears to improve detection of subtle or near invisible endometriosis in women with CPP and minimal visual findings at L/S and may serve to elevate diagnostic accuracy for endometriosis at laparoscopy.

Loss of peritoneal integrity provides a new paradigm to understand and treat chronic pelvic pain in women with mild forms of endometriosis and can be easily detected using intraoperative instillation of dye at the time of laparoscopy.

Endometriosis is a  common disease affecting 40 to 70% of reproductive-aged women with chronic  pelvic pain (CPP) and/or infertility. The purpose of this ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Sentinel Lymph Node Mapping and Biopsy for Endometrial Cancer at Early Stage with Laparoscopy

Sentinel lymph node (SLN) mapping and biopsy is a promising technique for visualizing and evaluating lymph node status in cancer. This approach has been recommended for low-risk endometrial cancer (EC) patients by authoritative international guidelines, but it has not been performed broadly in China and worldwide. This work aims to describe detailed SLN mapping and biopsy procedures to promote the clinical application. SLN mapping and postoperative pathologic ultrastaging were conducted in a patient with low-risk EC using indocyanine green (ICG) dye to track the SLNs under laparoscopy and resecting them completely for ultrastaging. In conclusion, this protocol describes details of ICG injection, and SLN mapping and biopsy in EC patients based on the experiences gained during clinical practice.

This protocol describes the identification and resection of sentinel lymph nodes to make the operation as easy and minimally invasive as possible.

Sentinel lymph node (SLN) mapping and biopsy is a promising technique for visualizing and evaluating lymph node status in cancer. This approach has been ...

Nanodrug Targeting Oncogenic miRNA-10b for Treating Metastatic Breast Cancer

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or excision. Many of these nodules are identified and excised via minimally invasive thoracic surgery; however, subcentimeter and subsolid nodules are frequently difficult to identify intra-operatively. This can be mitigated by the use of electromagnetic transthoracic needle localization. This protocol delineates the step-by-step process of electromagnetic localization from the pre-operative period to the postoperative period and is an adaptation of the electromagnetically guided percutaneous biopsy previously described by Arias et al. Pre-operative steps include obtaining a same day CT followed by the generation of a three-dimensional virtual map of the lung. From this map, the target lesion(s) and an entry site are chosen. In the operating room, the virtual reconstruction of the lung is then calibrated with the patient and the electromagnetic navigation platform. The patient is then sedated, intubated, and placed in the lateral decubitus position. Using a sterile technique and visualization from multiple views, the needle is inserted into the chest wall at the prechosen skin entry site and driven down to the target lesion. Dye is then injected into the lesion and, then, continuously during needle withdrawal, creating a tract for visualization intra-operatively. This method has many potential benefits when compared to the CT-guided localization, including a decreased radiation exposure and decreased time between the dye injection and the surgery. Dye diffusion from the pathway occurs over time, thereby limiting intra-operative nodule identification. By decreasing the time to surgery, there is a decrease in wait time for the patient, and less time for dye diffusion to occur, resulting in an improvement in nodule localization. When compared to electromagnetic bronchoscopy, airway architecture is no longer a limitation as the target nodule is accessed via a transparenchymal approach. Details of this procedure are described in a step-by-step fashion.

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

The increased use of chest computed tomography (CT) has led to an increased detection of pulmonary nodules requiring diagnostic evaluation and/or ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Biology

Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction defects can be assessed by tracking methylene blue dye flow within the urinary system. Methylene blue dye is injected into the renal pelvis of perinatal embryonic kidneys and dye flow is monitored from the renal pelvis of the kidney through the ureter and into the bladder lumen after applying hydrostatic pressure. Dye accumulation will be evident in the bladder lumen of the normal perinatal urinary tract, but will be constrained between the renal pelvis and the end point of an abnormal ureter, if urinary tract obstructions occur. This method facilitates the confirmation of urinary tract junction obstructions and visualization of hydronephrosis and hydroureter. This manuscript describes a protocol for methylene blue dye injection into the renal pelvis to confirm urinary tract junction obstructions.

Methylene blue dye injection into the renal pelvis facilitates the assessment of urinary tract junction obstruction defects during mouse embryonic urinary tract development. Here, a protocol for methylene blue dye injection into the renal pelvis is described.

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction ...