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Nitrocellulose coated slides

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Nitrocellulose-coated slides are a type of laboratory equipment used for various scientific applications. They provide a suitable surface for the immobilization of biomolecules, such as proteins and nucleic acids, enabling researchers to conduct various analytical and diagnostic procedures.

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Lab products found in correlation

Genepix pro 7, by Molecular Devices (3 mentions) Irdye 680 fluorophore, by LI COR (3 mentions) Genepix al4200, by Molecular Devices (3 mentions) Automated slide stainer, by Agilent Technologies (3 mentions) Pixcell iie lcm system, by Arcturus (2 mentions) Mapix, by Innopsys (2 mentions) Dylight 800 conjugated secondary antibodies, by New England Biolabs (2 mentions) Innoscan 710 ir scanner, by Innopsys (2 mentions) Proteome profiler mouse adipokine array kit, by R&D Systems (1 mentions) Array pro analyzer, by Media Cybernetics (1 mentions) Array pro analyzer, by Meyer Instruments (1 mentions) Tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions) Powerscanner, by Tecan (1 mentions) Sypro ruby protein blot stain, by Thermo Fisher Scientific (1 mentions) Irdye 680, by LI COR (1 mentions) Catalyzed signal amplification system kit, by Agilent Technologies (1 mentions)

Spelling variants of this product

Nitrocellulose-coated glass slides (6 citations) Nitrocellulose slides (5 citations) 16-pad nitrocellulose-coated slides (2 citations) Nitrocellulose-coated microscope slides (2 citations) 2 × 8 pad nitrocellulose-coated microarrays slides (2 citations)

16 protocols using nitrocellulose coated slides

1

Multiplexed Immunoassay Profiling of Tissue

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Enriched epithelial-cell subpopulations were isolated from 8 μM cryosections (>95% purity) using an Arcturus Pixcell IIe LCM system (Arcturus). Resultant cell lysates were printed in triplicate spots (approx. 10nL per spot) onto nitrocellulose-coated slides (Grace Biolabs) using an Aushon 2470 Arrayer (Aushon Biosystems) as described (18 (link)). Antibodies used on the arrays were extensively validated before use and are listed in Supplementary Table S1. RPPA immunostaining was performed as previously described (19 (link)).
For the calibration study, 20 breast cancers with varying degrees of known HLA-DR or HLA-DR/DP/DQ/DX expression from the nonimmunotherapy-treated TMA cohort were analyzed by identical methods to the above and a linear correlation was established against mIF percent positivity, yielding a cut-off point of 17,000 units comparable with 5% tumor-cell positivity.
Gonzalez-Ericsson P.I., Wulfkhule J.D., Gallagher R.I., Sun X., Axelrod M.L., Sheng Q., Luo N., Gomez H., Sanchez V., Sanders M., Pusztai L., Petricoin E., Blenman K.R, & Balko J.M. (2021). Tumor-Specific Major Histocompatibility-II Expression Predicts Benefit to Anti–PD-1/L1 Therapy in Patients With HER2-Negative Primary Breast Cancer. Clinical Cancer Research, 27(19), 5299-5306.
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2

NSCLC Cell Protein Profiling

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Antibody validation and RPPA analysis were performed as described previously [47 (link)]. Briefly, protein lysate was collected from NSCLC cell cultures after 72 hours in 150nM dasatinib or vehicle control. Protein lysates (1 μg/μL) were printed (2470 Arrayer; Aushon Biosystems, Burlington, MA) on nitrocellulose-coated slides (Grace Bio-Labs, Bend, OR) and immunostaining was performed using a DakoCytomation-catalyzed system and diaminobenzidine colorimetric reaction. Spot intensities were quantified using MicroVigene software (VigeneTech Inc., Carlisle, MA).
Peng S., Sen B., Mazumdar T., Byers L.A., Diao L., Wang J., Tong P., Giri U., Heymach J.V., Kadara H.N, & Johnson F.M. (2015). Dasatinib induces DNA damage and activates DNA repair pathways leading to senescence in non-small cell lung cancer cell lines with kinase-inactivating BRAF mutations. Oncotarget, 7(1), 565-579.
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3

Reverse Phase Protein Array Analysis of Adipokines

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Protein lysates were prepared by the BCM Antibody-Based Proteomics Core for reverse phase protein array assays. The Aushon 2470 Arrayer (Aushon BioSystems) with a 40 pin (185 μm) configuration was used to spot lysates onto nitrocellulose-coated slides (Grace Bio-Labs). The slides were probed with 220 antibodies against total and phospho-proteins using an automated slide stainer (Dako). Primary antibody binding was detected using a biotinylated secondary antibody followed by streptavidin-conjugated IRDye 680 fluorophore (LI-COR). Fluorescent-labeled slides were scanned on a GenePix AL4200 and the images were analyzed with GenePix Pro 7.0 (Molecular Devices). Background-subtracted total fluorescence intensities of each spot were normalized for variation in total protein (Sypro Ruby) and nonspecific labeling. For adipokine analysis, tissue lysates from four samples were pooled (125 μg/sample) and incubated with membranes from the Proteome Profiler Mouse Adipokine Array Kit (R&D Systems #ARY013) according to the manufacturer’s instructions.
Cox A.R., Masschelin P.M., Saha P.K., Felix J.B., Sharp R., Lian Z., Xia Y., Chernis N., Bader D.A., Kim K.H., Li X., Yoshino J., Li X., Li G., Sun Z., Wu H., Coarfa C., Moore D.D., Klein S., Sun K, & Hartig S.M. (2022). The rheumatoid arthritis drug auranofin lowers leptin levels and exerts anti-diabetic effects in obese mice. Cell metabolism, 34(12), 1932-1946.e7.
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4

Quantitative Protein Profiling Using RPPA

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Cell lysates, serial dilutions of standards, and positive and negative controls were arrayed on nitrocellulose-coated slides (Grace Bio-Labs) using the Quanteriz 2470 Arrayer. Each slide was probed with a validated primary antibody plus a biotin-conjugated secondary antibody. Signal detection was amplified using an Agilent GenPoint staining platform and visualized by DAP colorimetric reaction. The slides were scanned, analyzed, and quantified to generate spot intensity using customized software (Array-Pro Analyzer, Media Cybernetics). Each dilution curve was fitted with a logistic model (RPPA SPACE developed at MD Anderson). The protein concentrations were then normalized for protein loading. The corrections factor was calculated and normalized across sets via replicates-based normalization using an invariant set of control samples to adjust for batch differences between identical controls.26 (link)
Bridges C.S., Chen T.J., Puppi M., Rabin K.R, & Lacorazza H.D. (2022). Antileukemic properties of the kinase inhibitor OTSSP167 in T-cell acute lymphoblastic leukemia. Blood Advances, 7(3), 422-435.
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5

Reverse Phase Protein Array Analysis of Human Tumors

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RPPA data were generated at MD Anderson. Briefly, protein was extracted using RPPA lysis buffer (1% Triton X-100, 50 nmol/L Hepes (pH 7.4)), 150 nmol/L NaCl, 1.5 nmol/L MgCl2, 1 mmol/L EGTA, 100 nmol/L NaF, 10 nmol/L NaPPi, 10% glycerol, 1 nmol/L phenylmethylsulfonyl fluoride, 1 nmol/L Na3VO4, and 10 Ag/mL aprotinin) from human tumors and RPPA was performed as described previously20 (link),21 (link),50 (link),51 (link). Tumor lysates were adjusted to 1 μg/μL concentration, diluted in fivefold serial dilutions with lysis buffer, and printed on nitrocellulose-coated slides (Grace Bio-Labs, Bend, OR). Slides were probed with 191 validated primary antibodies followed by corresponding secondary antibodies (goat anti-rabbit IgG, goat anti-mouse IgG, or rabbit anti-goat IgG). Signal was captured using diaminobenzidine colorimetric reaction and normalized by using the fitted curve (“supercurve”) approach53 (link). In total, 255 samples were included in the analysis.
Oh S.C., Sohn B.H., Cheong J.H., Kim S.B., Lee J.E., Park K.C., Lee S.H., Park J.L., Park Y.Y., Lee H.S., Jang H.J., Park E.S., Kim S.C., Heo J., Chu I.S., Jang Y.J., Mok Y.J., Jung W., Kim B.H., Kim A., Cho J.Y., Lim J.Y., Hayashi Y., Song S., Elimova E., Estralla J.S., Lee J.H., Bhutani M.S., Lu Y., Liu W., Lee J., Kang W.K., Kim S., Noh S.H., Mills G.B., Kim S.Y., Ajani J.A, & Lee J.S. (2018). Clinical and genomic landscape of gastric cancer with a mesenchymal phenotype. Nature Communications, 9, 1777.
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6

Quantitative Protein Profiling of Epithelial Subpopulations

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Enriched epithelial cell subpopulations were isolated from 8um cryosections (>95% purity) using an Arcturus Pixcell IIe LCM system (Arcturus, Mountain View, CA, USA). Resultant cell lysates were printed in in triplicate spots (approx. 10nL per spot) onto nitrocellulose coated slides (Grace Biolabs, Bend, OR, USA) using an Aushon 2470 Arrayer (Aushon Biosystems, Billerica, MA, USA) as described (19 ). Antibodies used on the arrays were extensively validated before use and are listed in Supplementary Table 1. RPPA immunostaining was performed as previously described(20 (link)).
For the calibration study, twenty breast cancers with varying degrees of known HLA-DR or HLA-DR/DP/DQ/DX expression from the non-immunotherapy treated tissue microarray cohort were analyzed by identical methods to the above and a linear correlation was established against mIF percent positivity, yielding a cut point of 17,000 units comparable to 5% tumor cell positivity.
Gonzalez-Ericsson P.I., Wulfkhule J.D., Gallagher R.I., Sun X., Axelrod M.L., Sheng Q., Luo N., Gomez H., Sanchez V., Sanders M., Pusztai L., Petricoin E., Blenman K.R, & Balko J.M. (2021). Tumor-Specific Major Histocompatibility-II Expression Predicts Benefit to Anti–PD-1/L1 Therapy in Patients With HER2-Negative Primary Breast Cancer. Clinical Cancer Research, 27(19), 5299-5306.
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7

Reverse-phase Protein Array Analysis Protocol

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Reverse-phase protein arrays (RPPA) were analyzed and antibodies validated by the RPPA Core Facility at MD Anderson [43 (link)]. Total protein lysates were prepared by resuspending cells in lysis buffer (1% Triton X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM NaPPi, 10% glycerol, 1 mM PMSF, 1 mM Na3VO4, and 10 μg/mL aprotinin). Protein lysates were adjusted to a 1 μg/μL concentration, and a serial dilution of five concentrations was printed on nitrocellulose-coated slides (Grace Bio-Labs, Bend, OR) with 10% of the samples replicated for quality control (2470 Arrayer; Aushon Biosystems, Billerica, MA). Immunostaining was performed using a DakoCytomation-catalyzed system with the diaminobenzidine colorimetric reaction (Dako, Carpenteria, CA). Slides were scanned on a flatbed scanner to produce 16-bit tiff images. Spot intensities were analyzed and quantified using an Array-Pro Analyzer (Meyer Instruments, Houston, TX) to generate spot signal intensities. Overall, 285 unique antibodies and 4 secondary antibody negative controls were analyzed. The Gene Cluster 3.0 (http://cluster2.software.informer.com/3.0/) software was used for data analysis, and the heat map was created by Java TreeView (http://rana.lbl.gov/EisenSoftware.htm) software.
Liu W., Chen J., Tamayo A.T., Ruan C., Li L., Zhou S., Shen C., Young K.H., Westin J., Davis R.E., Hu S., Medeiros L.J., Ford R.J, & Pham L.V. (2017). Preclinical efficacy and biological effects of the oral proteasome inhibitor ixazomib in diffuse large B-cell lymphoma. Oncotarget, 9(1), 346-360.
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8

Quantitative Protein Array Analysis

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Cell lysates were prepared and analyzed as previously described (Teo et al., 2018 (link)). Briefly, medium was removed from the plates and cells were rinsed twice with PBS. Cells were lysed on ice for 20 minutes with occasional shaking every 5 minutes. Cell lysates were collected with a scraper and clarified by centrifugation at 18,000 rcf. for 10 min at 4°C. Clarified supernatants in biological triplicate were adjusted to 2 mg/mL concentration and printed onto nitrocellulose-coated slides (Grace Bio-Labs) in a dilution series (four serial 2-fold dilutions) in technical triplicate using an Aushon 2470 arrayer (Aushon Biosystems). Slides were blocked, probed with validated primary antibodies and detected with DyLight 800-conjugated secondary antibodies (New England BioLabs). Slides were read using an InnoScan 710-IR scanner (Innopsys) and quantified using Mapix (Innopsys). Relative fluorescence intensities were normalized to respective FastGreen-stained spots (total protein), and data were computationally analyzed as previously described (Byron, 2017 (link); Teo et al., 2018 (link)).
Gangoso E., Southgate B., Bradley L., Rus S., Galvez-Cancino F., McGivern N., Güç E., Kapourani C.A., Byron A., Ferguson K.M., Alfazema N., Morrison G., Grant V., Blin C., Sou I., Marques-Torrejon M.A., Conde L., Parrinello S., Herrero J., Beck S., Brandner S., Brennan P.M., Bertone P., Pollard J.W., Quezada S.A., Sproul D., Frame M.C., Serrels A, & Pollard S.M. (2021). Glioblastomas acquire myeloid-affiliated transcriptional programs via epigenetic immunoediting to elicit immune evasion. Cell, 184(9), 2454-2470.e26.
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9

RPPA Analysis of HNSCC Cell Cultures

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RPPA analysis and antibody validation were performed as previously described (31 (link)). Briefly, protein lysate was collected from subconfluent HNSCC cell cultures after 24 hours in full-serum medium (10% FBS). Protein lysates were adjusted to a 1-μg/μL concentration, and a serial dilution of 5 concentrations was printed, with 10% of the samples replicated for quality control (2470 Arrayer; Aushon Biosystems, Burlington, MA) on nitrocellulose-coated slides (Grace Bio-Labs, Bend, OR). Immunostaining was performed using a DakoCytomation-catalyzed system and diaminobenzidine colorimetric reaction. Spot intensities were analyzed and quantified using MicroVigene software (VigeneTech Inc., Carlisle, MA) to generate spot signal intensities.
Mazumdar T., Byers L.A., Shing Ng P.K., Mills G.B., Peng S., Diao L., Fan Y.H., Stemke-Hale K., Heymach J.V., Myers J.N., Glisson B.S, & Johnson F.M. (2014). A Comprehensive Evaluation of Biomarkers Predictive of Response to PI3K Inhibitors and of Resistance Mechanisms in Head and Neck Squamous Cell Carcinoma. Molecular cancer therapeutics, 13(11), 2738-2750.
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10

Quantifying ERBB2 Inhibition in Tumors

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To identify changes in ERBB2-mutant and wild-type tumors in response to ERBB2 inhibition, we used reverse-protein phase array (RPPA) analysis. Cellular proteins derived from the tumors were arrayed on nitrocellulose coated slides (Grace BioLabs, Bend, OR) by an Aushon 2470 Arrayer (Aushon BioSystems, Billerica, MA). Each slide was probed with a validated primary antibody with a Pearson correlation coefficient between RPPA and western blotting of greater than 0.7. Each dilution curve was fitted with a logistic model (“Supercurve Fitting” developed by the Department of Bioinformatics and Computational Biology in MD Anderson Cancer Center, http://bioinformatics.mdanderson.org/OOMPA).
Adam L., Lucas F.A., Fowler R., Yu Y., W.e.n.h.u.i.W.u., Liu Y., Wang H., Menter D., Tetzlaff M.T., Ensor J J.r., Manyam G., Arold S.T., Huff C., Kopetz S., Scheet P, & Overman M.J. (2018). DNA Sequencing of Small Bowel Adenocarcinomas Identifies Targetable Recurrent Mutations in the ERBB2 Signaling Pathway. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(2), 641-651.
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