Cells on 25 mm poly-L lysine-coated glass coverslips were rinsed twice with PBS, pH 7.2-7.4, fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, rinsed three times with PBS, incubated with PBS containing 0.3% Triton X-100 for 30 min, blocked in 10% goat serum in PBST for 1 hr, incubated with primary monoclonal anti-GFAP mouse antibody (Sigma, St. Louis, MO, USA) overnight at 4°C, rinsed three times with PBS, incubated with secondary goat anti-mouse Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) for 2 hr at RT, rinsed three times with PBS, mounted with glycerin, and examined under confocal microscope (Leica, USA) or inverted fluorescence microscope IX51 (Olympus, Japan).
Secondary goat anti mouse alexa fluor 488
Manufactured by Thermo Fisher Scientific
Sourced in United States
The secondary goat anti-mouse Alexa Fluor 488 is a fluorescently labeled secondary antibody used in various immunoassay techniques. It binds to mouse primary antibodies, allowing for the detection and visualization of target proteins or analytes.
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2 protocols using secondary goat anti mouse alexa fluor 488
1
Immunostaining of Astrocytes with GFAP
2
Quantifying Toxoplasma Infection and Replication
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For plaque assays, 500 freshly syringe-lysed intracellular parasites were allowed to invade a confluent monolayer of HFFs grown in 12-well plates in 500 µM IAA or vehicle. Six days post-infection, the infected monolayers were stained with crystal violet stain to determine host cell lysis as previously described (43) .
For doubling assays, purified intracellular parasites were allowed to invade confluent HFFs for 30 min. After invasion, infected HFFs were gently washed with culture medium to remove extracellular tachyzoites and fresh media was added. After 24 hrs, infected monolayers were fixed with 4% paraformaldehyde for 10 min at room temperature, incubated in blocking buffer for 30 min, then stained with α-SAG1 antibody (Invitrogen) for 1 hr at room temperature. The fixed cells were then washed with PBS and incubated with secondary goat anti-mouse Alexa-fluor 488 (Invitrogen) for 1 hr at room temperature. Cells were mounted with ProLong Gold Antifade Mounting solution (Invitrogen) containing DAPI. The number of parasites in 100 randomly selected vacuoles was counted at the designated time point (44) .
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
For doubling assays, purified intracellular parasites were allowed to invade confluent HFFs for 30 min. After invasion, infected HFFs were gently washed with culture medium to remove extracellular tachyzoites and fresh media was added. After 24 hrs, infected monolayers were fixed with 4% paraformaldehyde for 10 min at room temperature, incubated in blocking buffer for 30 min, then stained with α-SAG1 antibody (Invitrogen) for 1 hr at room temperature. The fixed cells were then washed with PBS and incubated with secondary goat anti-mouse Alexa-fluor 488 (Invitrogen) for 1 hr at room temperature. Cells were mounted with ProLong Gold Antifade Mounting solution (Invitrogen) containing DAPI. The number of parasites in 100 randomly selected vacuoles was counted at the designated time point (44) .
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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