For immunofluorescence staining, cells were grown on coverslips and cultured in the supplemented media at 37°C in an atmosphere of 5% CO2. Cells were fixed with ice-cold methanol for 10 min, after which excess methanol was removed. After three subsequent washes with PBS, cells were blocked and permeabilized for 1 hour with PBS containing 1% BSA, 5% goat serum and 0.05% Triton X-100 (Sigma–Aldrich, St. Louis, Missouri, USA). The cell coverslips were incubated with primary antibody (mouse anti-GHR B10 (Santa Cruz, CA, USA), mouse anti-PrlR, clone 1A2B1 (Invitrogen, Carlsbad, CA, USA) and rabbit anti-PrlR M-170 (Santa Cruz, CA, USA) overnight at 4°C, washed again 3 times with PBS, and incubated for 1 h at room temperature in the dark with the secondary antibodies alexa fluor 488 goat anti-mouse and alexa flour 594 goat anti-rabbit (both from Invitrogen, Carlsbad, CA, USA). Finally, the slides were washed, mounted with Vectashield containing DAPI (H-1200, Vector Laboratories, CA, USA) and stored at 4°C in the dark. Controls for specificity and background staining were performed by incubating with mouse IgG or rabbit IgG antibodies. Image acquisition was performed by confocal laser scanning microscopy (CLSM).
Alexa flour 594 goat anti rabbit
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594 goat anti-rabbit is a fluorescently labeled secondary antibody designed for the detection of rabbit primary antibodies in various immunoassay applications. It is a conjugate of a goat-derived anti-rabbit antibody and the Alexa Fluor 594 fluorescent dye.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (3 mentions)
Alexa flour 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Vectashield containing dapi h 1200, by Vector Laboratories (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Goat serum, by Merck Group (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mounting medium, by Thermo Fisher Scientific (1 mentions)
Tcs sp2 se confocal microscope, by Leica (1 mentions)
Ab140915, by Abcam (1 mentions)
Confocal microscope, by Leica (1 mentions)
Proliferating cell nuclear antigen (pcna), by Thermo Fisher Scientific (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Ab8069, by Abcam (1 mentions)
Ab189524, by Abcam (1 mentions)
6 protocols using alexa flour 594 goat anti rabbit
1
Immunofluorescence Staining of GHR and PrlR
2
Immunofluorescence Staining of Lamin A/C
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Twenty-four hours after siRNA treatment against lamin A/C protein, cells were fixed with 4% paraformaldehyde. Cells were permeabilized by incubation with 0.3% Triton X-100. Cells were incubated in a blocking serum in PBS with 5% donkey serum (017-000-121, Jackson Immuno Research Laboratories, West Grove, PA, USA). Primary antibody solutions were incubated on the cells for 1 h at 37 °C, followed by secondary antibody incubation of either Alexa Flour 594 goat anti-rabbit (Invitrogen, Waltham, MA, USA) or Alexa Fluor 647 donkey anti-mouse. For nuclear staining, cells were incubated with NucBlue Hoechst stain (Thermo Fisher Scientific, Waltham, MA, USA). For actin staining, cells were incubated with Alexa Fluor 488–phalloidin (Life Technologies, Carlsbad, CA, USA). Primary and secondary concentrations were both 1:300.
3
Immunocytochemistry for Astrocytes and Neurons
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For immunocytochemistry (ICC), astrocytes and neurons were seeded separately in 8-well chambered slide with a density of 10,000 and 25,000 cells per well, respectively. For co-culture staining astrocyte and neuron were seeded in 2:1 ratio. The cells were fixed with 4% paraformaldehyde (PFA). Blocking and permeabilization were done with 4% bovine serum albumin (BSA) (Cat. #A9418, Sigma-Aldrich, USA) and 0.3% Triton-X 100 for 1 h at room temperature (RT). Primary antibodies of EphrinA3 (Cat. #ab64814, RRID: AB_2246429, 1:1000, Abcam, Cambridge), ephA4 (Cat. #ab5389, RRID:AB_304853, 1:1000, Abcam, Cambridge) and MAP2 (Cat. #MAB3418, RRID:AB_94856, 1:1000, Chemicon, USA, Cat. #ab5392, 1:500, RRID:AB_2138153, Abcam, Cambridge) were incubated at 4 °C overnight whereas GFAP (Cat. #MAB360, RRID:AB_11212597, 1:1000, Millipore, USA) was incubated at RT for 1 h. Cells were then labelled with secondary antibodies, Alexa Fluor 488 goat anti-mouse and Alexa Flour 594 goat anti-rabbit (Cat. #A-11001, RRID:AB_2534069, #A-11037, RRID:AB_2534095, Invitrogen, USA) and goat anti-Chicken Cy3 (Cat. #ab97145, RRID:AB_10679516, 1:1000, Abcam, Cambridge) for 1 h at RT. The cells were mounted with hardest 4′,6-diamidino-2-phenylindole (DAPI; Cat. #H-1500, RRID:AB_2336788, Vector Labs, USA). Images were taken across the slide using AxioObserver.Z1 Apotome (Carl Zeiss, Germany).
4
Bag-1 and Beclin 1 Colocalization Assay
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Cells were seeded as 1 × 105 cells per well in 12-well plate containing poly-L-lysine coated coverslips, and co-transfected with His6Bag-1S or His6Bag-1L and His6Beclin 1 plasmid. 48 h post-transfection, culture medium was discarded, and cells were washed with phosphate buffered saline (PBS) solution. Cells were fixed in pre-chilled 4% paraformaldehyde and incubated for 10 min at room temperature, then, washed three times with PBS. Cells were blocked by 1 h incubation in blocking solution (3% BSA, 0.1% Triton-X 100 in PBS) and were incubated with appropriate primary antibodies overnight at 4 °C. Primary antibodies used were mouse anti-Bag-1 (1:200), rabbit anti-Beclin 1 (1:200). Following washing, cells were incubated for 1 h at RT with the secondary antibody (Alexa Flour® 488 goat anti-mouse or Alexa Flour® 594 goat anti-rabbit; 1:300 for both, Invitrogen, Carlsbad, CA, USA). After extensive washing, coverslips were mounted on slides using ProLong Diamond Antifade mounting medium containing DAPI (P36962, Thermo Fisher, Waltheim, MA, USA). Confocal images were obtained via a Leica TCS SP2 SE confocal microscope (Leica, Buffalo Grove, IL, USA).
5
Immunofluorescence Staining of Paraffin-Embedded Tissues
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The paraffin-embedded sections were deparaffinized and rehydrated, which was followed by antigen retrieval in boiling citrate buffer for 30 min. The sections were surrounded with a hydrophobic pen and rinsed with PBS. The sections were then permeabilized with 0.1% Triton X100 for 5 min in goat serum. The sections were blocked with 10% goat serum for 1 h and then incubated overnight at 4 °C with mouse anti-ATX (ab140915, Abcam, Waltham, MA, USA), mouse anti-lysophosphatidic acid (#504B3, Echelon Biosciences Inc., Logan, UT, USA), anti-phospho-ERK (#9101, Cell Signaling Technology, Inc. Waltham, MA, USA), PCNA (#13-3900, Invitrogen, Waltham, MA, USA), and anti-smooth muscle actin (#ab5694, Abcam, Waltham, MA, USA) as primary antibodies. The sections were then incubated with AlexaFluor 488 goat anti-mouse (#A28175, ThermoFisher Scientific, Waltham, MA, USA) and AlexaFlour 594 goat anti-rabbit (#A11012, ThermoFisher Scientific, Waltham, MA, USA) before then being rinsed with PBS. After 1 h, the sections were rinsed with PBS three times, mounted with a vector-shield mounting medium with DAPI (#H-1500, Vector Laboratories, Newark, CA, USA), and visualized using a Leica confocal microscope.
6
Immunofluorescence Labeling of Cell Markers
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The fixed cultures were washed three times with PBS before blocking and permeabilization with blocking buffer (1 % bovine serum albumin and 1 % Tween 20 in PBS) at room temperature for 1 h. For the labelling, different antibodies were used. To label CD44 on JIMT-1 cells, the primary rabbit anti-CD44 antibody (ab189524, 1:300) (Abcam, Cambridge, United Kingdom) was used. HDFs in mono- and co-cultures were stained with the primary mouse anti-vimentin antibody (ab8069, 1:500) (Abcam, Cambridge, United Kingdom). Vimentin was used to distinguish JIMT-1 cells from the HDFs in co-cultures. The samples were incubated overnight at 4 °C with the primary antibody followed by washing three times with PBS, and incubation for 2 h at room temperature with the secondary antibody Alexa Flour™ 594 goat anti-rabbit (A11037, 1:600) (ThermoFisher Scientific, Waltham, MA, USA) or Alexa Flour™488 goat anti-mouse (A11029, 1:1000) (ThermoFisher Scientific, Waltham, MA, USA). Then, the cultures were washed three times with PBS and the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/mL PBS) (ThermoFisher Scientific, Waltham, MA, USA) for 2 min at room temperature.
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