For analyzing hypoxic cells, tumor cells were incubated for 1.5 h with 150 µM Pimonidazol at 37 °C. Pimonidazol is only active in hypoxic cells and stabilizes covalent bound thiolgroups in proteins, peptides and amino acids. After preparing cytospins, tumor cells were stained with secondary Hypoxyprobe-RedAPC antibody over night at 4 °C. Slides were washed with PBS and mounted with Vectashield mounting medium including DAPI (Vector laboratories, Burlingame, CA, USA).
Vectashield mounting medium including dapi
Manufactured by Vector Laboratories
Sourced in United States
Vectashield mounting medium is a high-performance anti-fade reagent designed for fluorescence microscopy. It is formulated to preserve the brightness and stability of fluorophores. The product includes DAPI, a nuclear counterstain that labels DNA and can be used to visualize cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Dmire2, by Leica camera (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Volocity, by PerkinElmer (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Brdu assay kit, by Roche (1 mentions)
Gcdca, by Merck Group (1 mentions)
Tcdca, by Merck Group (1 mentions)
Pd98059, by Cayman Chemical (1 mentions)
Ag1478, by Merck Group (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
Pannoramic midi slide scanner, by 3DHISTECH (1 mentions)
Nycodenz, by Axis-Shield (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Rabbit anti fasn, by Abcam (1 mentions)
5 protocols using vectashield mounting medium including dapi
1
Hypoxia detection in tumor cells
2
Fluorescent Labeling of Bacteria for Phagocytosis
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Bacteria were labeled with fluorescein isothiocyanate (FITC) for the phagocytosis assay. 50 ml of cultured cells (mid-log phase, OD600nm = 2) were harvested by centrifugation. The cell pellet was washed with phosphate-buffered saline (PBS) and re-suspended in 1 ml of carbonate/bicarbonate buffer (pH 9.5, 0.1 M Na2CO3 and 0.2 M NaHCO3). The FITC (Sigma Chemical Co., St. Louis, Mo.) was dissolved in dimethyl sulfoxide at 10 mg/ml. 100 μl of this solution was added to 500 μl of the bacterial suspension. The mixture was rotated at 180 rpm for 1 h at room temperature. Bacteria were then washed twice in PBS to remove the unbound fluorochrome and re-suspended in 500 μl of HBSS (Hanks Balanced Salt Solution). Aliquots were prepared, frozen and stored in the dark until used. Aliquots were thawed just before use. Dilutions of stock suspensions of FITC-labeled bacteria or control bacteria were plated on agar to determine the number of CFU/ml. Coverslips were mounted with Vectashield mounting medium including DAPI (Vector Laboratories, Burlingame, CA). Staining was viewed with an inverted confocal microscope (DMIRE2, Leica) equipped with a 63XNA 1.4 objective lens using the LS 3D software (Leica). Images were analyzed using the ‘MetaMorph’ (Molecular Devices, Downington, PA) and Volocity (Perkin Elmer, version 6.0.1) software.
3
Isolation and Characterization of Mouse Hepatic Stellate Cells
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Isolation of primary mHSC was performed via pronase–collagenase perfusion followed by density gradient centrifugation in 13.2% Nycodenz (Axis-Shield PoC, Oslo, Norway) [33 (link)]. Purity of preparation was assessed by confirmation of vitamin A autofluorescence.
Isolated HSCs were allowed to attach for 2 h and were then stimulated with bile salts CDCA, GCDCA, TCDCA, and UDCA (Sigma-Aldrich, Darmstadt, Germany) for the time periods indicated, in the absence or presence of AG1478 (Sigma-Aldrich, Darmstadt, Germany) and PD98059 (Cayman, Ann Arbor, MI, USA). To quantify total DNA as a surrogate of cell number, HSCs were incubated with PicoGreen® (Invitrogen, Carlsbad, CA, USA) and fluorescence signals were detected with a CytoFluor 4000 system (PerSeptive Biosystems, Framingham, MA, USA). Proliferation of HSC was quantified using a BrdU-assay kit (Roche, Penzberg, Germany) according to the manufacturer’s instructions. To quantify total cell count, HSCs, seeded in Lab-Tek II Chamber Slides (Nunc, Rochester, NY, USA), were mounted on cover slides with Vectashield mounting medium including DAPI (Vector, Burlingame, CA, USA). Slides were scanned with a Pannoramic Midi Slide Scanner (3DHistech, Budapest, Hungary) and nucles count was performed with ImageJ2 software on the complete slide (0.7 cm2).
Isolated HSCs were allowed to attach for 2 h and were then stimulated with bile salts CDCA, GCDCA, TCDCA, and UDCA (Sigma-Aldrich, Darmstadt, Germany) for the time periods indicated, in the absence or presence of AG1478 (Sigma-Aldrich, Darmstadt, Germany) and PD98059 (Cayman, Ann Arbor, MI, USA). To quantify total DNA as a surrogate of cell number, HSCs were incubated with PicoGreen® (Invitrogen, Carlsbad, CA, USA) and fluorescence signals were detected with a CytoFluor 4000 system (PerSeptive Biosystems, Framingham, MA, USA). Proliferation of HSC was quantified using a BrdU-assay kit (Roche, Penzberg, Germany) according to the manufacturer’s instructions. To quantify total cell count, HSCs, seeded in Lab-Tek II Chamber Slides (Nunc, Rochester, NY, USA), were mounted on cover slides with Vectashield mounting medium including DAPI (Vector, Burlingame, CA, USA). Slides were scanned with a Pannoramic Midi Slide Scanner (3DHistech, Budapest, Hungary) and nucles count was performed with ImageJ2 software on the complete slide (0.7 cm2).
4
Immunofluorescence Staining of Heart
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Paraformaldehyde- fixed (4%) and paraffin embedded heart sections were deparaffinized and rehydrated, antigen retrieval was performed using a sodium citrate treatment. Alternatively, serial cryosections (10 μm) immobilized on Superfrost Plus slides (Thermoscientific) were rehydrated in PBS, fixed in 4% PFA for 10 min. Permeabilization of cardiac tissues was performed with 0.2% Triton X-100 for 20 min. After blocking of non specific sites with 1% BSA, the primary antibodies were incubated o/n at 4 °C. After labeling with appropriate secondary antibodies, the sections were mounted in Vectashield mounting medium including DAPI (Vector Laboratories) and imaged by confocal microscopy.
5
Immunohistochemical Analysis of Hippocampal Markers
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Animals (n 5 3 per genotype) were perfused transcardially with 0.1 M PBS followed by 4% PFA in 0.1 M PBS. Brains were removed, postfixed overnight at 48C and cryoprotected with 30% sucrose in 0.1 M PBS for 4 days at 48C. Brains were rapidly frozen in powdered dry ice and sliced into 40 mm-thick sagittal sections on a cryostat. Freefloating sections containing the hippocampus were washed three times with 0.1 M PBS and blocked by incubation with blocking solution (0.2% triton and 2.5% bovine serum albumin in 0.1 M PBS) for 1 hr followed by overnight incubation with primary antibodies in blocking solution at 48C. The sections were rinsed four times in 0.1 M PBS and incubated in blocking solution containing the appropriate secondary antibody for 2 hr. Sections were rinsed four times with 0.1 M PBS and subsequently mounted in Vectashield mounting medium including DAPI as a nuclear dye (Vector Laboratories) on glass slides. The following primary antibodies were used mouse anti-GFAP (1:1.000, Sigma) and rabbit anti-FASN (1:1.000, Abcam). Secondary antibodies were Alexa fluor 568-conjugated goat anti-mouse and Alexa fluor 488conjugated goat anti-rabbit.
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