Immunofluorescence staining

Materials
	PBS++ (50 ml PBS; 25 μl 2.5M CaCl2 ; 1M 50 μl MgCl2)
	Permeabilization solution (PBS++; 10% FCS; 0.2% Saponin; alternatively 0.2%
	Triton X-100 may be used instead of saponin as a detergent)
	Blocking solution (PBS++; 10% FCS)
	First and second antibody
	Mounting medium
	Nail polish

Procedure

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• After fixation wash (3 x with PBS++) the cover slips in the 24 well/plates
• Add 300 μl/well permeabilization solution (10 min)
• Wash samples 3 x with PBS++
• Incubate samples with 300 μl/well blocking solution (5 min)
• Add dropwise 25 μl/well of the first antibody (diluted 1:100 to 1:400 in blocking solution) on the cover slip
• Inubate 1 h at RT
• Wash samples 3 x with PBS++
• Incubate samples with 300 μl/well blocking solution (5 min)
• Add on drops 25 μl/well of the fluorescent coupled second antibody (diluted 1:100 to 1:200 in blocking solution) on the cover slip
• Incubate for 45 min at RT in the dark
• Wash samples 3 x with PBS++
• Transfer cover slips on the glass slide (add mounting medium in between)
• After drying seal the cover slips with nail polish.