HFF cells were cultured on circular glass coverslips (Electron Microscopy Sciences) and infected with parasites for 24 h. For visualization of dense granule proteins in the PV, cultures were fixed in 4% paraformaldehyde and permeabilized for 10 min with 0.01% saponin (Sigma). All samples were blocked for 1 h with 10% FBS and incubated for 1 h with a 1:500 dilution of primary rabbit anti-HA tag MAb (Cell Signaling) and a 1:1,000 dilution of primary mouse anti-GRA2 MAb or anti-GRA5 MAb (Biotem, France). Preparations were washed 3 times with DPBS supplemented with Ca2+ and Mg2+ and incubated for 1 h at room temperature with a 1:1,000 dilution of goat anti-rabbit and goat anti-mouse IgG secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594, respectively. Samples were mounted in SlowFade gold antifade with DAPI (Life Technologies) and imaged with a Nikon A1R SI confocal microscope (Nikon, Inc.). PVs were located using differential interference contrast microscopy (DIC). Confocal images were processed using the FIJI program (109 (link)).
Circular glass coverslips
Manufactured by Electron Microscopy Sciences
Circular glass coverslips are flat, round pieces of glass used to cover and protect samples in various microscopy applications. They provide a thin, transparent surface for mounting and examining specimens under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Mouse laminin, by Thermo Fisher Scientific (3 mentions)
Tro19622, by Bio-Techne (3 mentions)
Poly l lysine (pll), by Merck Group (3 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (3 mentions)
Saponin, by Merck Group (2 mentions)
A1rsi confocal microscope, by Nikon (2 mentions)
Brainphys neuronal medium, by STEMCELL (2 mentions)
Neural tissue dissociation kit postnatal neurons, by Miltenyi Biotec (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Slowfade gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (1 mentions)
5 protocols using circular glass coverslips
1
Visualizing Toxoplasma Parasitophorous Vacuole
2
Neuron Isolation and Glioma Co-culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neurons were isolated from the brains of P1 NSG; Nlgn3WT or NSG;Nlgn3y/− animals using the ‘Neural Tissue Dissociation Kit - Postnatal Neurons’ (Miltenyi), and followed by the ‘Neuron Isolation Kit, Mouse’ (Miltenyi) per manufacturer’s instructions. After isolation, 300,000 neurons were plated onto circular glass coverslips (Electron Microscopy Services) pre-treated for 1-hour RT with poly-L-lysine (Sigma) and then 3 hours at 37°C with 5 μg/mL mouse laminin (Thermo Fisher). Neurons are cultured in BrainPhys neuronal medium (Stemcell Technologies) supplemented with 1x glutamax (Invitrogen), pen/strep (Invitrogen), B27 supplement (Invitrogen), BDNF (10ng/mL; Shenandoah), and GDNF (5ng/mL; Shenandoah), TRO19622 (5μM; Tocris), beta-mercaptoethanol (Gibco), and 2% fetal bovine serum. Half of the medium was replenished on DIV 1 and UFDU was added at 1μM. This was repeated at DIV 3. On DIV 5, half of the medium was replaced with serum-free in the morning. In the afternoon, the medium was again replaced with half serum-free medium containing 75,000 glioma cells expressing PSD-95-RFP. Glioma cells were cultured with neurons for 72 hours and then fixed with 4% PFA for 20 minutes at room temperature and stained for immunofluorescence analysis as described below.
3
Visualizing Immune Response to Parasite Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDM and BMDC were harvested, seeded onto circular glass coverslips (Electron Microscopy Sciences), and incubated overnight. BMDM and BMDC were then activated with 100 U/ml IFN-γ and 10 U/ml TNF-α for 6 h and infected for 45 min with parasites at an MOI of 4. The coverslips were washed in DPBS, and cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized with 0.1% saponin (Sigma), and blocked in 10% FBS. For visualization, cultures were incubated with mouse anti-GRA5 antibody (Ab) (TG 17.113 at 1:2,000; Biotem) and rabbit anti-Irgb6 Ab (1:1,000) (74 (link)), washed, and incubated with anti-mouse IgG(H+L) coupled to Alexa Fluor 568 and anti-rabbit IgG(H+L) coupled to Alexa Fluor 488 as secondary antibodies (Invitrogen). Coverslips were mounted in ProLong gold with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen) and imaged at ×63 magnification with a Nikon A1R SI confocal microscope (Nikon, Inc.). All images were processed using the FIJI program (109 (link)). A minimum of 500 PVs of each strain was scored for quantification of Irgb6 coating of the PVM, as previously described (27 (link)).
4
Neuron Isolation and Glioma Co-culture
5
Synaptic Puncta Assay in Neuron-Glioma Co-culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For synaptic puncta assays, neurons were isolated from CD1 (The Jackson Laboratory) at P1 using he Neural Tissue Dissociation Kit -Postnatal Neurons (Miltenyi), and followed by the Neuron Isolation Kit, Mouse (Miltenyi) per manufacturers instructions. After isolation 200,000 neurons were plated onto circular glass coverslips (Electron Microscopy Services) pre-coated with poly-L-lysine (Sigma) and mouse laminin (Thermo Fisher) as described previously (Venkatesh). Neurons were cultured in BrainPhys neuronal medium containing B27 (Invitrogen), BDNF (10ng ml -1 , Shenandoah), GDNF (5ng ml -1 , Shenandoah), TRO19622 (5µM; Tocris), β-mercaptoethanol (Gibco). The medium was replenished on days in vitro (DIV) 1. On DIV 5, fresh media was added containing 50,000 glioma cells expressing PSD95-RFP. The neuron-glioma culture was incubated for 72hours and then fixed with 4% paraformaldehyde (PFA) for 20min at room temperature and stained for immunofluorescence analysis. DNA fragments above were stitched together using Gibson Assembly and transformed.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!