The primary mesencephalic and glial cultures were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 20 min and processed for immunocytochemical staining, as described previously (Ghosh et al., 2010 (link)). First, nonspecific sites were blocked with 2% bovine serum albumin, 0.5% Triton X-100 and 0.05% Tween-20 in PBS for 45 min at RT. Cells were then incubated overnight at 4°C with different primary antibodies such as TH (1:1600, mouse monoclonal; EMD Millipore, Billerica, MA) and GFAP (1:1000, mouse monoclonal; EMD Millipore). Appropriate secondary antibodies (Alexa Fluor 488 or 594 or 555; Invitrogen) were used followed by incubation with 10 μg/ml Hoechst 33342 (Invitrogen) for 5 min at RT to stain the nucleus. Coverslips containing stained cells were washed twice with PBS and mounted on poly-D-lysine-coated slides (Sigma, St Louis). Images of cells were captured with a SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI) attached to an inverted fluorescence microscope (model TE-2000U; Nikon, Tokyo, Japan).
Poly d lysine coated slides
Manufactured by Merck Group
Sourced in Canada
Poly-D-lysine-coated slides are a type of laboratory equipment designed to facilitate cell attachment and growth in in vitro cell culture experiments. The slides are coated with the positively charged amino acid polymer poly-D-lysine, which enhances the adhesion of various cell types to the surface of the slide.
Automatically generated - may contain errors
Lab products found in correlation
Rhodamine linked goat anti mouse igg1, by Santa Cruz Biotechnology (2 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Te2000 u, by Nikon (1 mentions)
Anti dsp, by Santa Cruz Biotechnology (1 mentions)
Anti ly75, by Santa Cruz Biotechnology (1 mentions)
Anti apc2, by Abcam (1 mentions)
Anti axin1, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 labeled goat anti rabbit antibody, by Abcam (1 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 or 594, by Thermo Fisher Scientific (1 mentions)
Inverted fluorescence microscope, by Nikon (1 mentions)
Alexa fluor 488 labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lsm 510 confocal microscope system, by Zeiss (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
4 protocols using poly d lysine coated slides
1
Immunocytochemical Staining of Primary Mesencephalic and Glial Cultures
2
Immunostaining of Cell Signaling Proteins
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Cells were plated on poly-d -lysine coated slides (Sigma Aldrich Oakville, ON, Canada), then fixed in 4% paraformaldehyde and permeabilized with 1 x PBS–0.2% Triton X-100. After blocking, cells were incubated with different primary antibodies, including anti-β-catenin (Santa-Cruz Biotechnology Dallas, TX, USA), anti-LY75 (Santa-Cruz Biotechnology Dallas, TX, USA and Abcam, Branford, CT, USA), anti-APC2 (Abcam, Branford, CT, USA), anti-Axin1 (Santa-Cruz Biotechnology Dallas, TX, USA), and anti-DSP (Santa-Cruz Biotechnology Dallas, TX, USA), and subsequently incubated with secondary antibodies, including rhodamine-linked goat-anti-mouse IgG1 (Santa Cruz Biotechnology Dallas, TX, USA) or Alexa Fluor 488-labeled goat anti-rabbit antibody (Abcam, Branford, CT, USA). Cells were finally stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a Zeiss LSM 700 confocal microscope (Carl Zeiss Meditec AG Jena, Germany).
3
Immunocytochemical Staining of Primary Mesencephalic Neurons
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The primary mesencephalic neurons were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min and processed for immunocytochemical staining. First, nonspecific sites were blocked with 2% bovine serum albumin, 0.5% Triton X-100, and 0.05% Tween 20 in PBS for 30 min at room temperature. Cells were then incubated with primary antibody such as anti-TH (1:500, mouse monoclonal) at 4°C overnight. Appropriate secondary antibodies (Alexa Fluor 488 or 594; Invitrogen) were used, followed by incubation with 10 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) for 5 min at room temperature to stain the nucleus. Cover slips containing stained cells were washed twice with PBS and mounted on poly-d -lysine–coated slides (Sigma-Aldrich). Cells were viewed under a Nikon inverted fluorescence microscope, and images were captured with a SPOT digital camera.
4
Immunostaining of Cellular Adhesion Proteins
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Cells were plated on poly-D-lysine coated slides (Sigma) and were fixed in 4% paraformaldehyde and permeabilized with Triton 1% X-100. After blocking, cells were incubated with primary antibodies, anti–E-cadherin (Abcam, 1:50) and anti-N-cadherin (Abcam, 1:50), subsequently with Rhodamine-linked goat-anti-mouse IgG1 (Santa Cruz, 1:50) and with Alexa Fluor 488–labeled secondary antibody (Life technologies, 1:500) respectively, and finally stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a Zeiss LSM 510 confocal microscope system.
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