Fixation and immunocytochemistry (fluorescence) in PFF-treated cultures
Maria Iuliano
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Abstract
This protocol outlines methods used for the preparation of in vitro models for fluorescent imaging including primary rat and mouse hippocampal cultures treated with PFFs.
Before start
Please read safety guidelines ahead of working with PFFs
Steps
Fixation and Immunocytochemistry (Fluorescence)
In a cell culture hood remove media and wash briefly with 1x PBS.
20rpm Fix using 4% PFA 4% sucrose in 1xPBS with gentle rocking/shaking.
20-30rpm Wash 1x PBS. Repeat 3 times.
20-30rpm Block in blocking buffer (5% NGS, 0.3% Triton-X in 1x PBS)
20rpm Incubate in solution containing primary antibodies diluted in 5% NGS in 1x PBS
20-30rpm Wash 1x PBS. Repeat 3 times.
20rpm Incubate in solution containing secondary antibodies diluted in 5% NGS in 1x PBS
20-30rpm Wash 1x PBS. Repeat 3 times.
Mount coverslips onto microscope slides using
Let cure 48h 0m 0s before imaging
