Paraffin-embedded lung sections from patients with PAH at lung transplant and control samples from unused donor lungs were provided by Dr. R. James White in the Department of Pulmonary and Critical Care Medicine. Normal lung tissue slides were purchased from Novus Biologicals (NBP2-30182). Informed consent to use the tissue for research purposes was previously obtained. Samples were deidentified. Lungs from both patients with idiopathic PAH (5 subjects) and healthy donors (unused donor lungs without evidence for pulmonary disease from 6 individuals) were studied.
Nbp2 30182
Manufactured by Novus Biologicals
The NBP2-30182 is a laboratory equipment product manufactured by Novus Biologicals. It is designed for scientific research and analysis purposes. The core function of this product is to perform specific tasks within a laboratory setting, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
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3 protocols using nbp2 30182
1
Pulmonary Hypertension Lung Tissue Analysis
2
Immunohistochemistry for EGR1 in Lung Tissue
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We performed immunohistochemistry using ImmPRESS HRP Horse Anti-Rabbit IgG PLUS Polymer Kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. We deparaffinized the paraffin-embedded slides in xylene and rehydrated them in a graded series of ethanol. We performed antigen retrieval by autoclaving the slides in 10 mM citrate buffer, at pH 6.0. We blocked the activity of the endogenous peroxidase with 3% hydrogen peroxide for 30 min. After blocking, we incubated the slides with a 1:200 dilution of rabbit monoclonal anti–EGR1 antibody (ab194357, Abcam, Cambridge, UK) at 4°C overnight in a humidified chamber. Next, we incubated the slides with ImmPRESS HRP horse anti-rabbit IgG polymer reagent (Vector Laboratories) for 30 min, followed by 3,3′-diaminobenzidine (DAB) staining for 40 s. We counterstained the slides with Mayer hematoxylin, dehydrated in a graded series of ethanol followed by xylene, and mounted with Mount Quick (Daido Sangyo, Saitama, Japan). Paraffin-embedded normal lung sections were purchased from Novus Biologicals (62-years old, male, NBP2-30182).
3
SARS-CoV-2 in Lung Tissue Analysis
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Formalin-fixed and paraffin-embedded (FFPE) lung tissue of 2 patients with confirmed SARS-CoV-2 infection (C21-20, C19-20) were used. As control tissue, FFPE lung of a healthy, 62-years old, male donor was used (NBP2-30182, Novusbio, cat. No 0028000B).
For the detection of Gene-specific RNA by in situhybridisation, the RNAscope 2.5 HD Reagent Kit-RED (code: 322350, Advanced Cell Diagnostics) and the probes (code: NCOA7 1029911-C1, ZBTB42 1029921-C1, OAS1 1029931-C1, ANKFY1 1029941-C1, UNC93B1 1029951-C1, SCARB2 1029961-C1; Advanced Cell Diagnostics) were designed (gene bank no: NM_001199622, NM_001137601, NM_016816, NM_016376, NM_030930, NM_005506) and purchased. As positive and negative control, a human ubiquitin and a plant probe were used, respectively (codes: 310041 and 310043, Advanced Cell Diagnostics). The protocol was followed according to the manufacturer's instructions. This work (ethics approval number 32077020.6.0000.0005) was approved on May 2020 by the National Committee in Ethics and Research, Brazil. in COMISSÃO NACIONAL DE ÉTICA EM PESQUISA.
For the detection of Gene-specific RNA by in situhybridisation, the RNAscope 2.5 HD Reagent Kit-RED (code: 322350, Advanced Cell Diagnostics) and the probes (code: NCOA7 1029911-C1, ZBTB42 1029921-C1, OAS1 1029931-C1, ANKFY1 1029941-C1, UNC93B1 1029951-C1, SCARB2 1029961-C1; Advanced Cell Diagnostics) were designed (gene bank no: NM_001199622, NM_001137601, NM_016816, NM_016376, NM_030930, NM_005506) and purchased. As positive and negative control, a human ubiquitin and a plant probe were used, respectively (codes: 310041 and 310043, Advanced Cell Diagnostics). The protocol was followed according to the manufacturer's instructions. This work (ethics approval number 32077020.6.0000.0005) was approved on May 2020 by the National Committee in Ethics and Research, Brazil. in COMISSÃO NACIONAL DE ÉTICA EM PESQUISA.
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