Formaldehyde-fixed and paraffin-embedded clinical tumor or HBV-infected liver samples were immunohistochemically analyzed for hepsin, HBx, and C3 on tissue microarray slides (Shanghai Outdo Biotech Co, China). These samples included eleven human distant normal liver tissues, twelve pairs of human HCC and peripheral non-tumor tissues, four pairs of normal liver tissues and hemangioma, and three pairs of peripheral normal tissues and hepatocirrhosis. Out of 49 total samples, 35 liver tissue samples contained HBV, as confirmed by patients' serum positive HBsAg. Patients' consent and approval by the local ethics committee were obtained for the use of clinical materials in this research. Immunohistochemical staining was performed as described previously [15 (link)].
Tissue microarray slides
Manufactured by Shanghai Outdo Biotech Co.
Sourced in China
Tissue microarray slides are a laboratory equipment used for the analysis and study of tissue samples. These slides contain multiple tissue samples arranged in a grid-like pattern, allowing for the simultaneous examination of various tissue types or samples. The core function of tissue microarray slides is to facilitate the efficient and parallel analysis of tissue specimens, enabling researchers to conduct comparative studies and high-throughput screening.
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Lab products found in correlation
Citrate buffer, by Beyotime (1 mentions)
Anti ddx5, by Santa Cruz Biotechnology (1 mentions)
Alexafluor 488 or 555 conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Confocal laser scanning microscopy, by Zeiss (1 mentions)
7 protocols using tissue microarray slides
1
Immunohistochemical Analysis of Liver Samples
2
DDX5 and O-GlcNAcylation in Colorectal Cancer
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For immunohistochemistry (IHC), human colorectal cancer tissue microarray slides (Outdo Biotech, Shanghai, China, #HColA180Su08). Patient information provided by the TMA supplier. After the tissue sections were deparaffinized and rehydrated, antigen retrieval was carried out in citrate buffer (Beyotime) at 100°C for 0.5 hour. Endogenous peroxidase was blocked with 3% peroxide for 15 minutes and blocked in buffer (5% BSA +0.1% Triton X‐100) for 1 hour and incubated overnight in primary antibody. The primary antibodies used were anti‐DDX5 (Santa Cruz, #sc‐166167, 1:300), anti‐O‐GlcNAcylation (Abeam, #ab2739, 1:100), and detection of signals using a Vectastain ABC kit (Vector Labs, Burlingame, CA, USA). For IF, cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS and blocking buffer (3% FBS +1% HISS +0.1% Triton X‐100), and then incubated overnight at 4°C in primary antibody. The primary antibodies used were anti‐DDX5 (Santa Cruz, #sc‐166167, 1:100), anti‐O‐GlcNAcylation (Abeam, #ab2739, 1:100). Fluorescent Alexa‐Fluor‐488 or ‐555‐conjugated secondary antibodies (Life technologies, Carlsbad, CA, USA) were used for detection.
3
Fluorescence In Situ Hybridization for circRNF10 and miR-934 in Breast Cancer
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The CY3-labeled circRNF10 probe and the FITC-labeled miR-934 probe were purchased from Geneseed Biotech (Guangzhou, China). For FISH in BC cells, MDA-MB-231 and BT-549 cells were cultured on coverslips, fixed with 4% formaldehyde, and permeabilized in PBS with 0.5% Triton X-100. FISH probes were diluted, denatured, balanced, and added to breast cancer cells overnight at 37 °C. For FISH in tissues, the tissue microarray slides (Outdo Biotech, Shanghai, China) were dewaxed in xylene and ethanol solutions. The subsequent procedure was similar to that described for FISH in cells. After hybridization, the nuclei were stained with DAPI. Confocal laser scanning microscopy (Zeiss, Jena, Germany) was then performed. The probe sequences were as follows: circRNF10-CY3 5′CY3-tacaaatgcgctcctagatgaat-3′CY3; hsa-miR-934-FITC 5′FITC-ccagtgtctccagtagtaga- 3′FITC. According to the cytoplasmic expression intensity of circRNF10, samples were classified as follows: negative or faint expression in most cells was defined as the negative group; moderate expression in <50% of cells or low expression in most cells was defined as the low expression group; and moderate to strong expression in most cells was defined as the high expression group.
4
Tissue Microarray Staining Intensity Scoring
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The tissue microarray slides were obtained from SHANGHAI OUTDO BIOTECH (Shanghai, China). Staining intensity was graded as follows: absent staining = 0, weak = 1, moderate = 2, and strong = 3. The percentage of staining was graded as follows: 0 (no positive cells), 1 (<25% positive cells), 2 (25%-50% positive cells), 3 (50%-75% positive cells), and 4 (>75% positive cells). The score for each tissue was calculated by multiplication; the range of this calculation was therefore 0 to 12 [52] (link).
5
Esophageal Squamous Carcinoma Tissue Microarray
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Tissue microarray slides were purchased from Shanghai Outdo Biotech Co., LTD, Shanghai, China. The slides included 95 esophageal squamous carcinoma specimens, 75 normal esophageal mucosa(NEM) tissue specimens. The detailed clinical-pathologic characteristics of patients with esophageal cancer are listed in Table 1 . All patients were clinically staged (TNM staging, tumor nodes metastasis staging) according to the seventh edition of the American Joint Committee on Cancer (AJCC) system for esophageal cancer14 (link). The pathological differentiated degrees are defined as follows: 1, High-differentiation carcinoma; 2, Medium-differentiation carcinoma; and 3, Low-differentiation. The degree of differentiation for the tumors in each of the patients was evaluated by two pathologists.
6
Breast Cancer Tissue Microarray Analysis
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Thirty pairs of paraffin-embedded breast carcinoma and adjacent normal breast tissue samples, as well as 112 paraffin-embedded breast carcinoma samples, were collected and these tissues were made into tissue microarray slides (Shanghai Outdo Biotech Co., Ltd). These samples were prospectively obtained from patients with breast cancer who underwent resection from January 2005 to December 2011 and were followed up for 2.1–11 years. The tissues larger than 5 cm from the tumor margin were selected and obtained as the adjacent normal tissues and these tissues were diagnosed and confirmed by pathologists as the normal tissues. Fresh tumor tissues were obtained from 40 patients with breast cancer undergoing neoadjuvant chemotherapy. All these patients received four cycles of AC (doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2) followed by four cycles of docetaxel (100 mg/m2). This study was approved by the Ethics Committee of the 940th Hospital of Joint Logistics Support Force of Chinese People’s Liberation Army.
7
Immunohistochemical Analysis of HCC
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Tissue sections in immunohistochemistry were enrolled at A liated Liutie Central Hospital of Guangxi Medical University and provided informed consent for tissue to be used for research studies. All tissues were snap-frozen and stored in liquid nitrogen immediately after surgical resection. Tissue microarray slides were purchased from Shanghai Outdo Biotech Co., Shanghai, China. The clinicopathologic features of the patients are described in Table 1. Each patient was diagnosed based on World Health Organization criteria, and tumor differentiation grade was classi ed according to Edmondson and Steiner (21) . Liver function was evaluated using the Child-Pugh scoring system. Tumor stages were classi ed based on the International Union against Cancer TNM classi cation system. OS was de ned as the time from surgery to death or surgery to last observation time. The OS data were obtained from the last follow-up for surviving HCC patients. Time to tumor recurrence was calculated from the surgical resection to the date of any relapse, including both extrahepatic metastasis and intrahepatic recurrence (22) . Male Balb/c-nude mice (four-week-old) were purchased from the Shanghai Institute of Material Medicine and raised under speci c pathogen-free conditions. The use of laboratory animals conformed to the NIH guide for Care and Use of Laboratory Animals, 8th ed.
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