Bacteria in midexponential growth phase were incubated overnight with 4× to 32× MIC of compound 1 . Following incubation, the cells were washed with PBS (3×), applied onto poly-l -lysine (Santa Cruz Biotechnology, Dallas, TX) coated microglass coverslips (Electron Microscopy Sciences, Hatfield, PA), and incubated for 15 min. The cells were fixed for 1 h with 2% glutaraldehyde, followed by a wash (3×) with sodium cacodylate buffer at pH 7.4. The samples were fixed with 1% osmium tetroxide for 1 h and rinsed (3×) in the buffer. The samples were then put through a graded ethanol series for dehydration, followed by critical point drying. The samples were then mounted on SEM stubs and sputter-coated with iridium. Microscopy and imaging were performed on a Magellan 400 XHR instrument (FEI, Hillsboro, OR).
Poly l lysine (pll)
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Poly-L-lysine is a synthetic polypeptide composed of the amino acid L-lysine. It is a positively charged polymer that can be used to coat surfaces and promote cell adhesion in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti p65 sc 372, by Santa Cruz Biotechnology (1 mentions)
Vectashield antifade mounting medium containing dapi, by Vector Laboratories (1 mentions)
Mouse anti β tubulin t8328, by Merck Group (1 mentions)
Microscope cover glasses, by Marienfeld (1 mentions)
β actin 66009 1 ig, by Proteintech (1 mentions)
Goat serum, by Santa Cruz Biotechnology (1 mentions)
Paraformaldehyde solution, by Santa Cruz Biotechnology (1 mentions)
Dab kit, by Santa Cruz Biotechnology (1 mentions)
Bicinchoninic acid bca protein assay kit, by Solarbio (1 mentions)
Qwin 500, by Leica (1 mentions)
5 protocols using poly l lysine (pll)
1
Scanning Electron Microscopy of Bacteria
2
Immunofluorescence Staining of p65 and β-Tubulin
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Microscope coverglasses (Marienfeld GmbH & Co. KG, Lauda-Königshofen, Baden-Württemberg, Germany) with a diameter of 12 mm were coated with 10% poly-l -lysine (Santa Cruz) in water and washed three times with phosphate buffered saline (PBS) in a 24-well plate before cells were seeded onto them. The cells were fixed for 15 min in 4% paraformaldehyde in PBS. The fixated cells were washed two times with PBS and then immersed in 0.5% Triton X-100 in PBS for 3 min to permeabilize the cells, followed by three washes with PBS. The cells were blocked using PBS with 5% normal donkey serum (NDS) overnight at 4 °C, followed by incubation with rabbit anti-p65 (sc372, Santa Cruz) and mouse anti-β-tubulin (T8328, Sigma) at a 1:100 dilution in PBS with 5% NDS for 1 h at 37 °C. After three washes with 0.05% Tween-20 in PBS (PBST), the cells were incubated with secondary antibodies conjugated with different fluorophores at a 1:200 dilution in PBS with 5% NDS for 30 min at room temperature. Finally, the cells were washed three times with PBST, and then the cover glass was transferred onto a small drop of VECTASHIELD antifade mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA) on a microscope slide. The cells were examined with a confocal LSM (laser scanning microscope) 710 microscope (Carl Zeiss, Oberkochen, Baden Württemberg, Germany).
3
Molecular Mechanisms of Liver Protection
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Yinchen, Zhizi, and Dahuang were provided by the particle pharmacy of Tianjin Medical University NanKai Hospital. Serum total bilirubin (TBIL) kits, direct bilirubin (DBIL) kits, alanine transaminase (ALT) kits, and aspartate aminotransferase (AST) kits were purchased from the National Institute of Biochemistry. Thermo Fisher Scientific (Shanghai, China) provided the Nrf2 antibody (PA5-27882), Keap1 antibody (PA5-99434), and protein marker (26617. Anti-NQO1 antibody (ab80588) and Anti-GST3/GST antibody (ab138491) were purchased from Abcam (Shanghai, China). β-Actin (66009-1-Ig) was purchased from Proteintech Group (Wuhan, China). Goat anti-rabbit immunoglobulin G (IgG) (ZB-2301) and anti-mouse IgG (ZB-2305) were purchased from Beijing Zhongshan Gold Bridge Biotechnology (Beijing, China). Poly (vinylidene fluoride) membrane (IPHV00010), electrochemiluminescence (ECL) kit (WBKLS0×500), and radioimmunoprecipitation assay buffer (RIPA) lysis buffer (20-188) were provided by Millipore Corporation (Shanghai, China). 4% paraformaldehyde solution, poly-L-lysine, antigen retrieval solution, goat serum, NOS3 (A-9; sc-376751), NOS2 (A-9; sc-7271), S-A/HRP, DAB kit, and hematoxylin and eosin staining kit were purchased from Santa Cruz Biotechnology (Beijing, China). Bicinchoninic acid (BCA) protein assay kit was provided by Beijing Solarbio Science & Technology (Beijing, China).
4
Culturing and Plating HeLa Cells
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HeLa cells (ATCC, CCL-2) were cultured in a growth medium (Dulbecco’s Modified Eagle Medium; DMEM) containing 10% fetal bovine serum and 1% penicillin–streptomycin in a cell culture flask (25 cm ) in an incubator at 37 C with 5% CO . Before the plating of cells in 8-well glass-bottom slides (µ-slide, ibidi), the wells were coated with poly-L-lysine (Santa Cruz Biotechnology, Dallas, TX, USA). After the addition of poly-L-lysine (0.1% (w/v)), the slides were incubated at 37 C for five minutes, washed with phosphate-buffered saline (PBS) twice, and dried at room temperature for three hours, followed by disinfection under ultraviolet (UV) light for 15 min. Subsequently, trypsinized cells were added at 100,000 cells per well (growth area per chamber: 1 cm ) and were incubated for 24 h before treatment with vibration.
5
Histopathological Analysis of Murine Lip
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Three mice from each group were sacrificed by cervical dislocation three and seven days after irradiation. The lower lip was dissected, fixed in 10% formalin and embedded in paraffin. Sections were cut at a thickness of 5 μm and stained with hematoxylin and eosin (H&E) for histopathological evaluation under the light microscope with ×400 magnification by pathologists. Other sections of 5 μm were placed on poly-L-Lysine coated slides and stained using mouse monoclonal antibodies to evaluate alpha-smooth muscle actin (α-SMA) expression (Santa Cruz Biotechnology, USA). Both epithelial thickness (µm) and area percent of cells expressing α-SMA were measured in 5 histological fields (×400) randomly captured in each slide with a digitized image analysis system using the software Leica Qwin 500.
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