The tissue sections were prepared as earlier described. The tissue was encircled with a PAP pen and blocked for 1 h with Ultra V Block (TA-060-UB, Thermo scientific). Approximately 25 µg antibody fragments were dissolved in Ultra V Block, 10 % goat serum and 1:100 anti-CK19 to a total volume of 100 µL and added to the encircled area. Incubation was performed for 3 h in humid chambers. The liquid was removed by aspiration, and the slide was washed four times 1 min in PBS. The slide was incubated for 30 min in the dark with mouse Cy3-conjugated anti-c-Myc antibody [9E10] 1:250 (Sigma-Aldrich), Alexa Fluor 488-conjugated goat anti-mouse IgG2a 1:500 (Invitrogen), DAPI 1:1000 (Invitrogen) and 10 % goat serum dissolved in Ultra V Block to 100 µL. Alternatively, the antibody fragments above were replaced with an anti-Ki67 antibody (Abcam) in a dilution of 1:100, and as secondary antibody, a goat anti-rabbit antibody coupled to Alexa 546 (Life technologies) was added in dilution of 1:500. The slide was washed three times 1 min in PBS and mounted with Fluoromount mounting media (Sigma-Aldrich) and cover glass.
Fluoromount mounting media
Manufactured by Merck Group
Sourced in United States
Fluoromount is a water-based, non-fluorescing mounting medium for microscopy. It is designed to preserve the fluorescence of stained specimens while providing a clear, durable mount.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Ultra 5 block, by Thermo Fisher Scientific (1 mentions)
Alexa 546, by Thermo Fisher Scientific (1 mentions)
Mouse cy3 conjugated anti c myc antibody 9e10, by Merck Group (1 mentions)
Anti ki67 antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Mc38 colon adenocarcinoma cell line, by Kerafast (1 mentions)
Rabbit anti piwi, by Santa Cruz Biotechnology (1 mentions)
Tritc conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Fitc conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Anti acetyl histone h4 antibody, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Sodium phosphate dibasic heptahydrate, by Merck Group (1 mentions)
Dapi 2 4 amidinophenyl indole 6 carboxamide dihydrochloride, by Tokyo Chemical Industry (1 mentions)
Trisodium citrate dihydrate, by Merck Group (1 mentions)
Indocyanine green, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Poly l arginine, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid edta, by Thermo Fisher Scientific (1 mentions)
6 protocols using fluoromount mounting media
1
Immunofluorescence Staining of Tissue Sections
2
Fluorescent Labeling and Imaging of Tumor Cells
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Murine B16F10 melanoma and LL2 Lewis lung carcinoma cell lines were purchased from ATCC. MC38 colon adenocarcinoma cell line was purchased from Kerafast (Boston, MA, USA). Cell lines were grown in Dulbecco’s modified eagle’s medium (DMEM) + 10% fetal bovine serum (FBS) media. All cell lines were maintained at low passage and routinely tested for mycoplasma. For Cy5 imaging, cells were resuspended and incubated with Cy5 labeled r9 or ACPP at 1 μM in 1% FBS. The cells were incubated for 2 h under rotation and covered to be protected from light at room temperature. Cells were then spun down (5 min at 2000 rpm) and resuspended in cell media for an additional 2 h. The cells were then fixed with 4% paraformaldehyde in phosphate buffered saline (PBS), washed twice with PBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI) stain (1:500). Finally, the cells were washed with PBS and mounted on slides using Fluoromount mounting media (Sigma, St Louis, MI, USA). Cells were imaged using a Keyence microscope with a 100× oil objective and high-resolution settings.
3
Ovarian Immunofluorescence Staining Protocol
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Ovaries were dissected in cold PBS and fixed in PBS with 4% paraformaldehyde for 15 min, then washed with PBT (PBS and 0.3% Triton X-100) five times for 15 min each. The ovaries were incubated in 0.5% goat serum diluted in PBT for 1 hr. Appropriate primary antibodies were added to PBS and incubated at 4° overnight, then washed with PBT five times for 15 min each. Appropriate secondary antibodies were then added and incubated at 25° for 2 hr; they were washed with PBT five times for 15 min each. After the last wash, the stained ovaries were mounted in Fluoromount mounting media (F4680; Sigma). Images were obtained with an inverted Zeiss LSM780 fitted with a UV laser.
The following primary and secondary antibodies were used: mouse monoclonal anti-Hts antibody 1B1 (DSHB, 1:100); rabbit anti-Piwi (1:200; Santa Cruz sc-98264); FITC-conjugated anti-mouse IgG (1:200; Jackson ImmunoResearch); and TRITC-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch).
The following primary and secondary antibodies were used: mouse monoclonal anti-Hts antibody 1B1 (DSHB, 1:100); rabbit anti-Piwi (1:200; Santa Cruz sc-98264); FITC-conjugated anti-mouse IgG (1:200; Jackson ImmunoResearch); and TRITC-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch).
4
Immunostaining of Larval Polytene Chromosomes
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The third-instar larval polytene chromosomes was dissected and immunostained36 (link). Chromosomes were incubated with mouse monoclonal anti-HP1a (HP1a C1A9, 1:100) or rabbit polyclonal anti-acetyl-histone H4 Antibody (EMD Millipore, 06-866, 1:200). After incubation for 1 h at room temperature, chromosomes were washed three times in PBS and incubated with a goat anti-mouse antibody conjugated to FITC (DSHB, 1:100) and a goat anti-rabbit antibody conjugated to FITC (DSHB, 1:100) for 2 h (room temperature). After one PBS wash, the slides were incubated with 4,6-diamidino-2-phenylindole (Sigma, 1: 1000) to stain DNA. The slides were mounted in Fluoromount mounting media (Sigma, F4680), and the immunostaining images were performed by using a Nikon ECLIPSE Ti inverted microscope (×100 model). Adobe Photoshop software was used for measurements.
5
Synthesis and Characterization of PLL-based Theranostic Nanoplatform
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PLL (Molecular weight = 120 kDa, ~574 lysine unit, one HBr per lysine residual) was procured from Polysciences (Warrington, PA, USA). Trisodium citrate dihydrate was procured from the Merck (Darmstadt, Germany). Sodium phosphate dibasic heptahydrate, fluoromount mounting media, and ICG were purchased from the Sigma Aldrich (St. Louis, MO, USA) and were used as received without further purification. Stock solutions of the ICG and salts were prepared in de-ionized (DI) water (18.2 MΩ Millipore, Sartorius system). All the prepared stock solutions were stored at 4 °C. Cancerous cells were procured from the National Centre for Cell Science (NCCS) Pune, India. Fetal Bovine Serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM), 0.25% trypsin-1 mM ethylenediaminetetraacetic acid (EDTA), penicillin-streptomycin, and 2.5% trypsin without phenol red were purchased from Gibco (Thermo Fisher Scientific Inc., India). Colorimetric assay (MTT, (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)) was procured from Himedia chemicals (India). For nucleus staining, DAPI (2-(4-amidinophenyl) indole-6-carboxamide-dihydrochloride) was procured from the Tokyo Chemical Industry (TCI, India).
6
Fluorescent Cellular Imaging Protocol
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Poly-l -arginine (PLA, MW > 70 000 Da, ∼365 arginine unit, one HCl per residue) and ethylenediaminetetraacetic acid tetrasodium salt hydrate (EDTA), indocyanine green (ICG) and fluoromount mounting media were purchased from Sigma Aldrich (St. Louis, MO, USA) and used without any further purification. The stock solutions of the chemicals were prepared in de-ionized (DI) water (Millipore 18.2 MΩ, Sartorius system). HeLa cells were procured from the National Centre for Cell Science (NCCS) Pune, India. Fetal Bovine Serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), penicillin–streptomycin, 0.25% trypsin-1 mM ethylenediaminetetraacetic acid (EDTA) and 2.5% trypsin without phenol red were procured from Gibco, Thermo Fisher Scientific. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was procured from Himedia (India).
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