Immunofluorescence Analysis of Hippocampal Synaptic Proteins
Corresponding Organization : University of Milan
Other organizations : Vita-Salute San Raffaele University
Variable analysis
- Antibody treatments (anti-GFP, anti-PSD95, anti-JIP1, anti-β-arrestin2, anti-JNK3)
- Immunofluorescence signal intensity and localization of target proteins
- Hippocampal primary cultures at DIV14
- Fixation in 4% paraformaldehyde (PFA) and 4% sucrose solution for 30 min
- Permeabilization with phosphate-buffered saline (PBS) at pH 7.4, containing 0.5% Triton X-100, for 3 min
- Blocking in PBS containing 1% BSA, 0.2% Triton X-100 for 1 h
- Incubation with primary antibodies in PBS containing 1% BSA and 0.2% Triton X-100 overnight at 4 °C
- Incubation with secondary antibodies (AlexFluor) for 1 h at room temperature
- Staining of nuclei with 2 mg/mL Hoechst
- Use of ProLong Glass Antifade Mountant as a mounting agent
- No positive or negative controls were explicitly mentioned in the protocol.
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