Sea block

Manufactured by Thermo Fisher Scientific

Sourced in United States

Sea Block is a lab equipment product from Thermo Fisher Scientific. It is designed for temperature control and sample incubation purposes in research and scientific applications. The core function of Sea Block is to maintain a stable and consistent temperature environment for samples during various laboratory procedures.

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Lab products found in correlation

Prolong antifade mountant, by Thermo Fisher Scientific (3 mentions) Ab34710, by Abcam (2 mentions) Sea block, by Abcam (2 mentions) Ab6586, by Abcam (2 mentions) Criterion tgx stain free gel, by Bio-Rad (2 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions) Bis tris page gel, by Thermo Fisher Scientific (2 mentions) Restore western blot stripping buffer, by Thermo Fisher Scientific (2 mentions) Tris glycine sds buffer, by Bio-Rad (2 mentions) Chemidoc touch system, by Bio-Rad (2 mentions) Chemidoc mp, by Bio-Rad (2 mentions) Criterion blotter system, by Bio-Rad (2 mentions) Trans blot turbo system, by Bio-Rad (2 mentions) Cytofix cytoperm fixation permeabilization kit, by BD (2 mentions) Alexa fluor 555, by Thermo Fisher Scientific (2 mentions) Dc2012, by Biocare Medical (2 mentions) Nitrocellulose membrane, by Cytiva (2 mentions) Lauryl maltoside detergent, by Abcam (2 mentions) Image studio light version 5.2, by LI COR (2 mentions) Chemidoc mp imaging system, by Bio-Rad (2 mentions)

Spelling variants of this product

Sea Block blocking buffer (34 citations) SEA block buffer (7 citations) Sea Block Blocking Solution (6 citations) PBS-SEA Block buffer (2 citations)

39 protocols using sea block

Immunofluorescent Characterization of Extracellular Matrix

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Paraffin-embedded sections were deparaffinized and rehydrated before antigen retrieval in a Decloaking Chamber™ NxGen (Biocare Medical, model no. DC2012) at 95 °C for 40 min. with antigen retrieval buffer (Abcam, #ab94674). Sections were rinsed with 1× Phosphate-Buffered Saline (PBS, Mediatech, #21-040), permeabilized with 0.05% TWEEN-20 (Sigma-Aldrich, #P9416) for 10 min., and then blocked with Sea Block (Fisher Scientific, #37527) for 30 min. before incubation with primary antibodies. Primary antibodies were diluted in Sea Block as follows: rabbit anti-collagen I at 1:100 (Abcam, #ab34710), rabbit anti-collagen IV at 1:500 (Abcam, #ab6586), and rabbit anti-laminin at 1:200 (Abcam, #ab11575). Sections were incubated with primary antibodies for 1 hr at room temperature, rinsed, and then incubated with secondary antibodies for 1 hr at 37 °C (goat anti-rabbit Alexa Fluor 488, Thermo Fisher, #A11034, diluted 1:300 in Sea Block). After secondary antibody incubation, sections were rinsed and mounted with Mowiol mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI) to stain for cell nuclei. Slides were imaged on a Nikon A1 Confocal Laser Microscope System.

Su J., Satchell S.C., Shah R.N, & Wertheim J.A. (2018). Kidney Decellularized Extracellular Matrix Hydrogels: Rheological Characterization and Human Glomerular Endothelial Cell Response to Encapsulation. Journal of biomedical materials research. Part A, 106(9), 2448-2462.

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Immunoblotting Analysis of DAP12 in NK Cells

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Immunoblotting was performed as described [108). Antibodies used for immunoblotting analysis were: monoclonal mouse/human α-DAP12 (D7G1X, Cell Signaling Technology) and mouse α-GAPDH (H86045M, Meridian Life Science). NK-cells were isolated by negative selection using Miltenyi Biotec NK-cell isolation kit (130-115-818) according to manufacturer’s instructions. 5x10⁵ cells were used for each well; Sham = 3 mice/sample, CLP = 9 mice/sample. Cells were then lysed with 2X lysis buffer (20mM Tris pH 8.0, 2mM EDTA, 2 mM Na₃VO₄, 20mM DTT, 2% SDS, and 20% glycerol) at 95°C for 5 min. Lysates were sonicated to reduce viscosity and loaded on 10–20% Tris-HCl Protein Gel (3450033, Bio Rad Criterion). Separated proteins were transferred to PVDF membranes (Millipore) and blocked for 1 hr in 1:1 PBS:SEA Block (Thermo-Fisher) IRDye 800CW or IRDye 680-conjugated secondary antibodies were diluted in SEA Block and incubated with PVDF membranes for 30 min at room temperature. Membranes were imaged using Licor Odyssey Infrared detector.

Jensen I.J., Winborn C.S., Fosdick M.G., Shao P., Tremblay M.M., Shan Q., Tripathy S.K., Snyder C.M., Xue H.H., Griffith T.S., Houtman J.C, & Badovinac V.P. (2018). Polymicrobial sepsis influences NK-cell-mediated immunity by diminishing NK-cell-intrinsic receptor-mediated effector responses to viral ligands or infections. PLoS Pathogens, 14(10), e1007405.

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Immunostaining of Neuronal Markers

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Neurons were fixed with 3.7% formaldehyde, permeabilized with 0.3% Triton X-100 in phosphate-buffered saline (PBS) at room temperature (RT), and blocked with 100% SeaBlock (Fisher Scientific, Ottawa, ON, Canada). Neurons were incubated overnight with primary antibody at 4 °C, then with secondary antibody at room temperature, and mounted using ProLong Gold (Fisher Scientific, Ottawa, ON, Canada. The following antibodies were used: rabbit anti-G3BP (abcam, ab214946 and ab217225, Toronto, ON, Canada), chicken anti-beta tubulin III (Aves Labs TUJ, Burlington, ON, Canada), rabbit anti-A1 (abcam ab197854), and donkey anti-chicken DyLight 405 (Jackson Immunoresearch 103-475-155, Burlington, ON, Canada).

Li M., Hamilton R., Salapa H.E, & Levin M.C. (2021). Pro-Inflammatory Cytokines and Antibodies Induce hnRNP A1 Dysfunction in Mouse Primary Cortical Neurons. Brain Sciences, 11(10), 1282.

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Immunofluorescence Staining Protocol

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To perform immunofluorescence staining, cell cultures were fixed with 4% PFA in PBS for 10 min, then washed with PBS twice before blocking in 1.5% SEA block (Thermo Fisher Scientific, 107452659) in TBST (0.1% Tween-20 in TBS). After minimally 30 min at room temperature, switched to primary antibody (diluted in blocking reagent) incubation in 4° overnight. After washing three times with TBST, switched to secondary antibody (diluted in blocking reagent) incubation for minimally 45 min in room temperature, blocking light. This was followed by three washes with TBST, then the cells were kept in PBS for confocal imaging. For freshly isolated cells (Figure 1—figure supplement 1H), cells were processed with Cytospin (Thermo Scientific) followed by the procedure described above. Quantification was performed with automatic program created in ImageJ.

Guo Q., Kim A., Li B., Ransick A., Bugacov H., Chen X., Lindström N., Brown A., Oxburgh L., Ren B, & McMahon A.P. (2021). A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells. eLife, 10, e64444.

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Western Blot Analysis of Shh-Signaling Proteins

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Lysates from 3T3-[Shh-BlastR;Cas9] cells were prepared in SDS sample buffer (50 mM Tris HCl pH 6.8, 8% v/v glycerol, 2% w/v SDS, 100 mM DTT, 0.1 mg/mL bromophenol blue), boiled and sonicated. Samples were loaded onto a 4–15% Criterion TGX Stainfree gel (Bio-Rad), and run for 25 min, 300V in Tris/Glycine/SDS buffer (Bio-Rad), before being transferred onto a PVDF membrane using a Transblot Turbo system (Bio-Rad). Membranes were blocked in 1:1 PBS:SeaBlock (Thermo Scientific) for 1 h at room temperature, and subsequently incubated with the indicated primary antibody for 16 h at 4 °C (Supplementary Table 10). After incubation with HRP-conjugated secondary antibody, blots were developed using Supersignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) and imaged on a ChemiDoc MP (Bio-Rad). Membranes were stripped using Restore Western Blot stripping buffer (Thermo-Fisher) and re-probed as described.
For analysis of immunoprecipitations, Western blotting was performed as described above, except samples were separated in 4–12% Bis-Tris PAGE gels (Invitrogen) using MOPS running buffer, transferred to PVDF membranes using the Criterion Blotter system (Bio-Rad), developed using ECL or ECL 2 chemiluminescence detection kits (Pierce), and imaged on a Chemidoc Touch system (Bio-Rad).

Breslow D.K., Hoogendoorn S., Kopp A.R., Morgens D.W., Vu B.K., Kennedy M.C., Han K., Li A., Hess G.T., Bassik M.C., Chen J.K, & Nachury M.V. (2018). A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies. Nature genetics, 50(3), 460-471.

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Immunohistochemical Analysis of γH2AX in Kidney Tissue

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PFA-fixed and paraffin-embedded kidney tissue sections (3 µm) were deparaffinized in Neoclear (Sigma-Aldrich, #109843, St. Louis, MO, USA) and rehydrated in a graded ethanol series (96%, 70% and 30% for 5 min each) to H₂O. Antigen retrieval was performed with Rodent Decloacer (Zytomed Systems GmbH, RD913L, Berlin, Germany) at 90–120 °C for 20 min. Peroxidase block was performed in 3% H₂O₂ for 10 min at room temperature. Afterwards, tissue was blocked (Sea Block, Thermo Scientific™, #37527, Waltham, MA, USA) at room temperature for 1 h. Primary antibody rabbit anti-mouse γH2AX (S139, γH2AX; Cell signaling, #2577, 1:1000, Danvers, MA, USA) was applied overnight at 4 °C. Following primary antibody incubations tissues section were treated with ZytoChem Plus HRP Polymer (Zytomed Systems GmbH, ZUC053, Berlin, Germany) for 30 min at room temperature. Tissue was then incubated in AEC permanent (Zytomed Systems GmbH, ZUC054, Berlin, Germany) for 5 min and subsequently counterstained with hematoxylin. Analysis was performed by light microscopy using 10 random fields of view per kidney section, for medulla and cortex separately. Quantification of γH2AX staining was performed by counting the percentage of γH2AX-positive nuclei relative to all nuclei per field of view.

Selle J., Bohl K., Höpker K., Wilke R., Dinger K., Kasper P., Abend B., Schermer B., Müller R.U., Kurschat C., Nüsken K.D., Nüsken E., Meyer D., Savai Pullamsetti S., Schumacher B., Dötsch J, & Alcazar M.A. (2023). Perinatal Obesity Sensitizes for Premature Kidney Aging Signaling. International Journal of Molecular Sciences, 24(3), 2508.

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FACS Analysis of Fixed Cells

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The Cytofix/Cytoperm^™ Fixation/Permeabilization Kit (BD Biosciences, Cat#554714) was used for fluorescence associated cell sorting analysis. 1×10⁶ cells were initially harvested and fixed in the Cytofix/Cytoperm solution for 20 minutes at 4°C. Cells were then washed with Perm/Wash (2x). Cells were incubated in primary antibody diluted in 1:1 mixture of SEA BLOCK (ThermoFisher Scientific, Cat#37527) and Perm/Wash overnight at 4°C on a mini-tube rotator. Samples were then washed with Perm/Wash (2x) and incubated in secondary antibody at room temperature for 1 hour. Samples were then washed with Perm/Wash (2x) and resuspended in ice-cold buffer (DPBS, 10% FBS, 10% Sodium-Azide). Samples were analyzed at the USC Stem Cell Flow Cytometry Facility.

Martirosian V., Deshpande K., Zhou H., Shen K., Smith K., Northcott P., Lin M., Stepanosyan V., Das D., Remsik J., Isakov D., Boire A., De Feyter H., Hurth K., Li S., Wiemels J., Nakamura B., Shao L., Danilov C., Chen T, & Neman J. (2021). Medulloblastoma uses GABA transaminase to survive in the cerebrospinal fluid microenvironment and promote leptomeningeal dissemination. Cell reports, 35(13), 109302.

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Immunofluorescence Staining of Muscle Samples

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Immunofluorescence and histological staining were performed as previously described 55. Gastrocnemius (GC) muscle samples were flash‐frozen in liquid nitrogen, 2‐methylbutane solution. Frozen sections (10‐μm) were fixed with 4% paraformaldehyde for 10 minutes. Sections were washed with phosphate‐buffered saline and permeabilized with 0.3% Triton X‐100 for 5 minutes, and then blocked with Seablock (Thermo Fisher Scientific, Waltham, MA) for 1 hour at room temperature. Cells or frozen sections were incubated at 4°C overnight with a primary antibody as follows: rabbit antidystrophin (1:200, Abcam, Cambridge, MA), antidystrophin‐PE (1:300, SC Biotechnology, Santa Cruz, CA), rabbit anti‐laminin 1 antibody (1:100, Abcam, Cambridge, MA), mouse anti‐Pax7 (1:100, DSHB, Iowa), or mouse antiembryonic myosin heavy chain (1:50, eMyHC, DSHB, IA). A Mouse on Mouse kit (Vector Laboratories, Burlingame, CA) was used per conditions according to the manufacturer's protocol. Secondary antibodies used were donkey anti‐rabbit or anti‐mouse Alexa 488‐ and Alexa 594‐conjugated IgG (Invitrogen and Jackson ImmunoResearch, West Grove, PA, respectively) per the manufacturer's instructions. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, 1:1,000, Invitrogen). Image acquisition was performed with a Leica Microsystems Inc. (Buffalo Grove, IL) camera at ×4–40 magnification.

Matre P.R., Mu X., Wu J., Danila D., Hall M.A., Kolonin M.G., Darabi R, & Huard J. (2019). CRISPR/Cas9‐Based Dystrophin Restoration Reveals a Novel Role for Dystrophin in Bioenergetics and Stress Resistance of Muscle Progenitors. Stem Cells (Dayton, Ohio), 37(12), 1615-1628.

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Western Blot Analysis of Cell Signaling Proteins

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Cell lysates were collected by rinsing samples with cold PBS, followed by a five minute lysis in mRIPA buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1% Triton, 1% Na-DOC, 0.1% SDS) with 1 mM EGTA, 1 mM Na3VO4, 10 mM Na4P2O7, and 1 mM PMSF (protease inhibitors). Cell lysates were separated via SDS-PAGE, transferred to PVDF membranes (Bio-Rad), and washed in Buffer A (25 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) + 4% SeaBlock (Thermo Fisher Scientific, Waltham, MA) overnight at 4°C. Membranes were incubated with anti-Vinculin (ab18058, Abcam), GAPDH (ab8245, Abcam), ERK2 (ab7948, Abcam), p130Cas (ab108320, Abcam), SORBS1 (ab4551, Abcam), SORBS3 (GTX-115362, Genetex), Filamin (ab51217, Abcam), or Paxillin (ab32084, Abcam) antibodies for 1 hour, washed with Buffer A containing SeaBlock, and incubated in streptavidin horseradish-peroxidase-conjugated secondary antibodies (Bio-Rad) for 30 minutes at room temperature. Immunoblots were visualized using ECL reagent (Pierce). All western blot antibodies were obtained from Abcam (Cambridge, England).

Holle A.W., McIntyre A.J., Kehe J., Wijesekara P., Young J.L., Vincent L.G, & Engler A.J. (2016). High content image analysis of focal adhesion-dependent mechanosensitive stem cell differentiation. Integrative biology : quantitative biosciences from nano to macro, 8(10), 1049-1058.

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Immunostaining and Imaging of Brain Sections

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Brains were serially sectioned at 30 μm using a Cryotome FSE and placed into a 12 well plate filled with PBS with about 14 sections per well. The wells were put in 10% SEA BLOCK (Thermo) for an hour on an orbital shaker at 60 rpm. Sections were moved into primary solution: mouse anti‐rabbit MAP2 (1:1000, Abcam) and rabbit anti‐mouse DARPP32 (1:1000, Abcam) and placed on a 4°C orbital shaker overnight. Sections were then washed with PBS three times and placed into secondary antibody stain consisting of 5% SEA BLOCK, AlexaFluor 488 anti‐mouse (1:1000, LifeTech), and AlexFluor 647 anti‐rabbit (1:1000, LifeTech) on a 4°C orbital shaker for an hour. After incubation, the wells were spiked with Hoescht (1:1000) for 5‐10 minutes on an orbital shaker, then transferred to phosphate buffered saline plus 1% Triton‐X (PBST) and washed three times. To utilize the autodetection software on the AxioScan, sections were immersed in 0.01% Sudan Black in 70% EtOH, gently agitated for 1 minute and then allowed to rest for 1 minute before being transferred into fresh PBS. The sections were then mounted onto microscope slides (Thermo Fisher Scientific) sequentially from the beginning to the end of the striatum and covered with a coverslip using Fluoromount (Sigma). Whole brain sections were then scanned and stitched together at 20× using a Zeiss AxioScan.

Dahlenburg H., Cameron D., Yang S., Bachman A., Pollock K., Cary W., Pham M., Hendrix K., White J., Nelson H., Deng P., Anderson J.S., Fink K, & Nolta J. (2021). A novel Huntington's disease mouse model to assess the role of neuroinflammation on disease progression and to develop human cell therapies. Stem Cells Translational Medicine, 10(7), 1033-1043.

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