The RA-h cell line, purchased from Shanghai SH Biotechnology Co., Ltd., was cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin/glutamine (PSG, Hangzhou Genom Biopharmaceutical Technology Co., Ltd.) and incubated at 37°C in 5% CO2. RA-h cells were identified by immunofluorescence, which involved adding 4% paraformaldehyde (1 mL/well) for 20 min to fix the cells, adding 0.5% Triton for 10 minutes, and blocking with 5% bovine serum albumin (BSA) for 1 h. The sections were incubated first with a monoclonal mouse anti-GFAP primary antibody overnight at 4°C (ab4648, Abcam) and then with an Alexa Fluor 488 (A-11001, Life Technologies, MA, USA, dilution 1 : 1000) secondary antibody for 1 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, R37606, Life Technologies), and coverslips with SlowFade Diamond Antifade Mountant (S36963, Life Technologies, Rockford, USA) were added. The mounted slides were observed using a microscope (DMI3000B, Leica).
R37606
Manufactured by Thermo Fisher Scientific
Sourced in United States
The R37606 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for general laboratory use. The core function of the R37606 is to provide a controlled environment for various laboratory experiments and processes.
Automatically generated - may contain errors
Lab products found in correlation
Bz x710, by Keyence (3 mentions)
A7906, by Merck Group (2 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Mouse anti beta 3 tubulin, by Merck Group (2 mentions)
Ab9610, by Merck Group (2 mentions)
Mab386, by Merck Group (2 mentions)
Rabbit anti neurofilament h, by Merck Group (2 mentions)
Smi 99p 100, by Merck Group (2 mentions)
Rabbit anti gfap, by Agilent Technologies (2 mentions)
Mouse anti active β catenin anti abc, by Merck Group (2 mentions)
Dmi3000b, by Leica (1 mentions)
Alexafluor488 a 11001, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Slowfade diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Ab4648, by Abcam (1 mentions)
A1r confocal microscope system, by Nikon (1 mentions)
Rabbit anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Ab203445, by Abcam (1 mentions)
23 protocols using r37606
1
Immunofluorescent Characterization of RA-h Cells
2
3D Cytoskeletal Organization Analysis
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F-actin and vinculin co-localisation was assessed in 2D, 2.5D and 3D co-cultures. After 72 h of incubation, cells were fixed in 4% paraformaldehyde and then washed twice in 0.05% Tween-20 in PBS wash buffer, before permeabilisation with 0.5% Triton X-100 solution in PBS blocked with 1% BSA in PBS. Then, cells were stained with mouse monoclonal anti-vinculin primary antibody (1:100 dilution, ab18058, Abcam) at 4 °C overnight. Following washing with wash buffer, spheroids were incubated with Alexa Fluor 488-conjugated secondary antibody (1:200 dilution, A11059, Rabbit Anti-Mouse IgG (H+L); Invitrogen, Carlsbad, CA, USA) and tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin (1:200, R415; Life Technologies Pty Ltd., Welshpool, Australia). Finally, cell nuclei were counterstained with DAPI (R37606, Life Technologies, and Grand Island, NY, USA) for 5 min and washed with wash buffer. Fluorescence images of the cells were captured with a laser scanning confocal microscope (LSCM) (objective 40×, excitation wavelengths: 488, 405 and 561 nm; A1R+ confocal microscope system; Nikon, Tokyo, Japan). Prior to imaging, spheroids were immersed in PBS to avoid drying out.
3
Profiling CXCL12 and Keratin-19 in FFPE Tumor Tissues
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FFPE human pancreatic and colorectal tumor arrays (US Biomax) were deparaffinized in xylene, washed three times with ethanol, and rehydrated in a serial concentration of ethanol (from 95 to 50%), and finally in water. For antigen retrieval, the sections were boiled in 10 mM Tris, pH 8.8 plus 1 mM EDTA for 10 min, followed by cooling down for 30 min, a wash with PBS, and blocking with 1% BSA/PBS at room temperature for 1 h. Following two washes with 0.05% Tween-20/PBS and one with PBS, Alexa Fluor 568-conjugated anti-KRT19 antibody (Abcam, ab203445) and FITC conjugated anti-CXCL12 antibody (R&D Systems, IC350F) were applied and the sections were incubated at room temperature for 1 h. Finally, the sections were stained with DAPI (Thermo Fisher, R37606) for 10 min and washed with 0.05% Tween-20/PBS for two times and once with PBS, followed by application of mounting medium (Thermo Fisher, P36961) and imaging with Leica SP8 confocal microscope. Images were analyzed and exported through ImageJ.
4
Cytotoxicity Screening of NSC Compounds
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The 5 × 103 cells/well of MCF-7 or MDA-MB-231cell-lines were seeded in 96 well plates. The next day, cells were treated with 0.16, 0.8, 4, and 20 µM of NSC 13030 , 24198 , 57774 , 137420 , or DMSO (Sigma-Aldrich–Cat D9170, St. Louis, MO, USA) as control. After 48 h of incubation, cells were stained with DAPI (Thermo Fischer–Cat R37606, Waltham, MA, USA) and images of the entire wells were taken with a 20× objective and a DAPI filter cube using Lionheart FX automated microscope. The image analysis and cell counts were done using Gen5 software. All conditions were done in triplicates.
5
Immunofluorescence Staining of Cells
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Cells were cultured in polymer dishes as described previously5 (link). Cells were fixed for 20 min using 4% (by weight) formaldehyde solution (Sigma-Aldrich 252,549). Subsequently, cells were permeabilized with 0.5% by volume Tween 20 (Sigma-Aldrich P1379) in PBS for 20 min, and then blocked with 1% by weight bovine serum albumin (BSA, Sigma-Aldrich A7906), 22.5 mg/mL glycine in PBST (0.1% by volume Tween 20 in PBS) for 30 min. Cells were incubated with the primary antibodies diluted in 1% BSA in PBST at 4 °C overnight. After that, cells were incubated with the secondary antibodies in 1% BSA at room temperature for 1 h. Finally, the nuclei were stained with DAPI or Hoechst (Thermofisher Scientific R37606 or R37605) fluorescent dyes. The primary antibody used: Atf2 (abcam ab32019), Beta-Catenin (abcam ab19381), GEF-H1 (abcam ab155785), Lef1 (abcam ab137872), Oct4 (abcam ab19857), RNA pol II (abcam ab5408), Smad4 (CST 46,535).
6
Immunostaining of Mouse Embryos
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Mouse embryos were usually fixed with 4% paraformaldehyde for 1 hour and then permeabilized with 0.5% Triton X-100 for 1 hour at room temperature. However, for the calculation of interpronuclear distance, geometric center distance, and diameters, the embryos were fixed and permeabilized using 4% paraformaldehyde containing 0.2% Triton X-100 for 20 min at room temperature (Figs. 2E and 6G and figs. S2A and S14F). The embryos were then blocked with 1% bovine serum albumin (Sigma-Aldrich) at room temperature for 1 hour and incubated with the primary antibodies against EHMT2 (1:400; Invitrogen, 435200) and H3K9me2 (1:400; Abcam, ab1220) at 4°C overnight. After washing three times with PBS containing 0.05% Tween 20, the embryos were incubated with the appropriate secondary antibodies at room temperature for 1 hour. The nuclei were stained with DAPI (Thermo Fisher Scientific, R37606). Images were captured under identical imaging conditions on the same confocal laser scanning microscope (Carl Zeiss LSM 880). Z projections were done by maximum intensity projections of the indicated Z-stacks. Quantitative fluorescence image analysis was performed using ImageJ (Fiji) software (https://imagej.net/software/fiji/downloads ). The intensity of fluorescence was calculated from images of stacked or sectioned images after the signal areas were detected automatically in regions of interest.
7
Immunolabeling of Glial Cell Markers
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To explore the distribution of immunoreactive glial cells in neurons, astrocytes, and microglias, double immunofluorescence staining of glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) were performed. For analyzing the viability of NA neurons in the LC, double immunofluorescence staining of dopamine β hydroxylase (DβH) was performed. Sections of 40-μm-thickness of the striatum, SNc, and LC were used. The slices were washed 3 times with PBS, followed by incubation with 10% normal horse serum and primary antibodies; rabbit anti-GFAP antibody (1:1000; Novus Biologicals, Littleton, CO, USA) and rabbit anti-Iba1 antibody (1:250; Wako Pure Chemical Industries, Osaka, Japan) and rabbit-anti DβH antibody (1:500; Sigma-Aldrich Co. LLC, St. Louis, MO, USA) for 24 h at 4 °C, respectively. After rinsing in PBS, sections were incubated for 1 h in FITC-conjugated affinity-purified donkey anti-rabbit IgG (H + L) and 4,6-diamidino-2-phenylindole (DAPI; 2 drops/mL, R37606; Thermo Fisher, Waltham, MA, USA) in a dark chamber. The sections were then extensively washed with PBS and coverslipped. Both TH and fluorescent immunoreactivities were visualized using an inverted fluorescence phase-contrast microscope BZ-X710 (Keyence, Osaka, Japan).
8
Immunofluorescence Assay for Myotube Morphology
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Immunofluorescence experiments and cell morphology measurements were performed using a previously described protocol.[44 ] The following antibodies were used: mouse anti‐MHC (1:2 MF20, R&D Systems, MAB4470) and anti‐mouse secondary antibody Alexa Fluor 568 conjugated (1:100, Invitrogen, A‐21144). The samples were washed three times and nuclei were counterstained with DAPI (Thermo Fisher Scientific, R37606). Myotube width was analyzed by ImageJ, as previously described.[25 ]
9
Immunostaining of Induced Pluripotent Stem Cells
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After 4–5 passages, the iPSCs were counted and 1–2 million cells were seeded on 24-well cell culture dishes pre-coated with 0.1% gelatin, and grown for 24–48 hr in 2i medium until 80% confluence. Cells were washed three times with DPBS (1×) and fixed with 4% paraformaldehyde at room temperature for 15 min. Cells were permeabilized by incubation with 0.2% Triton X-100 (Sigma-Aldrich, #T8787) and dissolved in 10% BSA (MPBIO, #0218054991) in 1× DPBS at room temperature for 30 min. Afterward, the permeabilized cells were washed twice with 1× DPBS and incubated with primary antibodies for NANOG (Novus, #NB100-58842, 1:500), OCT4 (Santa Cruz Biotechnology, #sc-5279, 1:500) and SOX2 (Santa Cruz; #sc-17320, 1:500) at 4°C overnight. The cells were then washed three times for 5 min with DPBS (1×) and incubated with secondary antibodies required for the respective primary antibody (donkey-anti-rabbit: Thermo Fisher Scientific #A24870, 1:250; rabbit-anti-mouse: Thermo Fisher Scientific #A21063, 1:500; donkey-anti-goat: Abcam #ab6949, 1:500) in darkness at room temperature for 2 hr. Cells were washed three times with DPBS (1×) for 5 min. The cells were further stained with 1× DAPI (Thermo Fisher Scientific, #R37606) and imaged with an Axio Vert.A1 (Zeiss).
10
AAV-Mediated CaMKII-GFP Expression
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AAV:CaMKII-GFP, as described above. After 2 weeks recovery, animal was perfused with 4% paraformaldehyde, followed by 24 h fixation in further 4% paraformaldehyde. The brain was cryoprotected in 30% sucrose before being sectioned (30 µm) on a cryostat. Sections were stained for DAPI (Thermo Fisher R37606, 5 min at room temperature) before mounting coverslips on slides in Fluoromount (Sigma F4680) and dried. Images were captured on the confocal microscope (Ti2, Nikon, Japan).
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