- Immunostaining protocol
=Immunofluorescence=
Coverslip preparation
* coat sterile coverslip with either Gelatine (0,1% in dH2O) or polyaminoacid poly-ornithine (0,1mg/ml; e.g. for Cerebella primary cell culture)poly-D-Lysine (0,1mg/ml, e.g. Cortex. Coat with polyaminoacid either in bulk or in plate* wash coverslips with dH2O or PBS if coating with polyamioacid and let dry!
* if necessary coat coverslips with Fibronectin (e.g. bovine plasma, Calbiochem) 50µg/ml in DMEM w/o serum, diluted from 1mg/ml stock 45min @ RT, gently rocking; wash 1x with DMEM
* seed cells in appropriate density the day before transfection (e.g. 1,5x105c/6well for HeLa; 1-1,5x105c/6well for NIH-3T3)
Transfection
* if needed, transfect cells using Exgen (1- max 3µg pDNA / 6well well; 8 equivalents = 13µl/well for 3µg DNA in 100µl 150mM NaCL/1ml +DMEM or 200µl NaCL/2ml +DMEM; spin 280rcf, 5min @ RT; change media after 4h til ON incubation)* wash cells 2x in either
PBS or TBSFixation
* Fixation of cells using 4% PFA (Formaldehyde ) in PBS/TBS or CBS (Cytoskeleton Buffer with Sucrose; if staining cytoskeletal filaments) for 5-20min @ RT; or using 100% -20°C Methanol 5-20min @ RT (Methanol will lead to denaturing of the protein)* Wash cells 2x with PBS/TBS
Staining
* For Immunostaining permeabilize the cells with 0.2-0.5% Triton-X 100) in TBS 20min @ RT (harsh) or 0.05-0.1% Saponin in PBS/TBS 20min @ RT (mild)
* Rinse with TBS-0,1%TX
* Block cells with either in Abdil (Antibody Dilution Solution 2% BSA) 10min @ RT or 5-10% Goat serum (in TBS) 60min @ RT; or Serum appropriate for secondary antibody (ideally serum of 2nd AB producing host).
* Incubate with 1st AB ON @ 4°C in Abdil (or PBS/0.1% Triton-X 100) (e.g. 1:50-1:1000). If labelling with more than one AB apply all together (if that does not work, one after another). Pipette 50-100µl directly onto cover slip and place piece of Parafilm on top; seal plate with Parafilm
* Wash 3x 10min with TBS-0.1%TX
* Incubate with 2nd AB in Abdil (or PBS/0,1% Triton-X 100 1h @ RT) (e.g. αRabbit-Cy3 1:200; Alexa-Flour-Dyes work well 1:500)
* Wash 3x 10min with PBS-0.1%TX (if counterstaining with Phalloidin or DAPI, proceed with staining after 2nd wash step)
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