Procedures
tumor, Dehydration
TUMOR, DEHYDRATION, STERILE, BLIND, PRIMARY TUMOR, DEHYDRATION
In the ICG group, ICG was endoscopically injected around the tumor in patients one day before operation; 1.25 mg/mL ICG (Dandong Yichuang Pharmaceutical Co) was prepared in sterile water, and 0.5 mL of the solution was injected into the submucosal layer at four quadrants around the primary tumor, amounting to 2.5 mg of ICG. We used the PINPOINT Endoscopic Fluorescence Imaging System (NOVADAQ, Stryker, US) equipped with a fluorescence mode to obtain NIR fluorescent images in the ICG group. A simple finger-click can convert visible light into NIR images (infrared imaging, green fluorescence, and color-segmented fluorescence) without the need to change any equipment. Intraoperatively, the fluorescent mode was switched according to the situation (Supplementary Fig. All the operations were performed by two surgeons (C.-H.Z. and C.-M.H.) who are members of the same surgical team. All the participating surgeons in our study met the following criteria: they had performed more than 100 laparoscopic radical gastrectomies, completed a learning curve in laparoscopic radical LN dissection, passed the blind surgical video examination, and had ample experience in ICG-guided LN dissection for GC. The surgeons were unaware of the specific allocation of the patient before the start of surgery, to prevent any potential discrimination of surgical strategy.All pathological evaluations were performed in a standard manner. For the pathological evaluation protocol, we referred to the LNs containing isolated tumor cells, defined as single tumor cells or small clusters of cells ≤0.2 mm in greatest diameter, without stromal reaction, are classified as pN0 in GCInformation regarding hematoxylin-eosin staining of paraffin sections includes the following steps:(1) Tissue embedding. (1) ethanol dehydration: tissues are dehydrated gradually with ethanol solutions of different concentrations (70%, 80%, 90%, 95%, 100%, and 100%) for 40 min each. (2) clearing: the tissues are immersed in three xylene baths, 1 h for each bath. (3) impregnation: the tissues are immersed in three paraffin baths, 1 h for each bath. (4) embedding: liquid paraffin is poured into a mold box, and the tissue block that has been impregnated with paraffin is laid flat on the bottom. Notably, the cutting surface should be placed facing downwards. After the paraffin has solidified, the embedding frame is removed. Once the tissue block has cooled down and becomes completely hard, the excess paraffin around the tissue is trimmed and kept moderately to facilitate sectioning.(2) Section preparation: the pre-cooled wax block is fixed onto the microtome, ensuring that the section of the wax block is parallel to the blade, which is typically tilted at 15°. The wheel advance mechanism is rotated and slice thickness is adjusted to 4 μm to obtain evenly thick slices. A brush is held in the left hand and the microtome handle is rotated with the right hand to cut the slices. The slices are gently lifted with the brush and the excess wax is tweezed. The slices are placed face up in the water bath of the slide warmer, which is set at a temperature of approximately 45 °C. After flattening the slices, they are picked with forceps. The slices are attached to the glass slides by immersing one end of a slide vertically in the water and the forceps are used to push the slice two-thirds of the way onto the slide. After attaching the slices to the slides, they are left to air dry and then placed in a slide warmer at 65 °C for 1 h, followed by a 2-h baking process in an oven.Deparaffinization of paraffin-embedded tissue sections: the paraffin sections are embedded in xylene I for 10 min, followed by xylene II for 10 min, and xylene III for 10 min. Then, the sections are gradually deparaffinized in a series of solutions: anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and finally 50% ethanol for 5 min.HE staining: the paraffin sections were stained with hematoxylin for 0.5–1 min, rinsed with tap water, differentiated in 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blue with 1% ammonia water for 1 min, rinsed with running water for a few seconds, and counterstained with eosin for several seconds, followed by rinsing with running water.(5) Dehydration and mounting of slides: the paraffin sections were sequentially immersed in 75% ethanol for 2 min, 85% ethanol for 2 min, and then in absolute ethanol for 5 min twice. Subsequently, the sections were cleared in xylene for 5 min and mounted with neutral gum after being removed from the xylene bath.(6) Interpretation of results: the cell nucleus is blue, and the cytoplasm is red.The extent of gastric resection and D2 lymphadenectomy was determined according to the tumor location, as indicated in the Japanese guidelines