Immunofluorescence Microscopy Staining Protocol with or without DAPI

Biochemistry, Molecular Biology, and Cell Biology Protocols >>  Immunofluorescence Microscopy Staining Protocol with or without DAPI

Solutions:
5x PB Buffer ( = 250 mM phosphate buffer, pH 7.2 after dilution)
To make 500mL, 
Mix 140mL 0.25M  NaH2P0
+ 360mL 0.25M NaH2P0
OR 
1x PB, 500mL
35 mL 0.2M NaH2P0
90 mL 0.2M NaH2P0
375mL water
1x PB is required for the procedures below

20% Triton
· Weigh 2g Triton on a balance by pipeting into a tube, 
· Make up to 10mL with distilled water 
· Rock overnight 

4% paraformaldehyde (STIR IN FUMEHOOD) 
To make 50 mL, 
· use ~40 mL water to dissolve 2g paraformaidehyde 
· add 200uL 0.2N NAOH and heat, while stirring in the fumehood 
· once dissolved, + 10mL of 5x PB buffer 
· freeze in ~10-15mL aliquots in the –20 freezer

Normal Goat Serum—NGS
· store in aliquots in the freezer

Notes: 
· if after fixing the cells, leave them wet in the fridge, covered with the PB from the wash
· be careful when pipeting and removing solutions; otherwise, cells will come off 

Procedure without DAPI
1. Wash 1 x with PB 
2. Fix with 4% paraformaldehyde (10 min., 15-30niin. if cells easily come off) 
3. Wash 3x with PB (5 min. each) 
4. Preincubation: for every 1 mL PB, + 1 00uL NGS, 10uL 20% Triton (15 min.) 
5. Primary antibody: for every 1mL total volume, add 20uL NGS, 5uL triton (2hrs); dilution of primary varies with each antibody and must be tested empirically or according to manufacturer's directions
6. Wash 3x with PB (10 min. each) 
7. Secondary antibody: choose the correct one (either goat anti-mouse, or goat anti-rabbit) CY3 (1/800 dilution): for every 1mL PB, 1.25uL secondary, 5uL triton, 25uL NGS (30 minutes-don't go too much longer, or else high background) 
8. Wash 3x with PB (15 min. each) 
9. Take out the mowial to thaw; take out cover slip, drain it a bit with Kleenex, +20uL mowial on the cells and cover the cover slip with it 
10. Invert the cover slip on the slide 
11. Place nail polish on the four corners of the cover slip. When the nail polish dries, put nail polish all around the slides of the cover slip. 
12. You may view it or put it in the dark at four degrees. The slides may be stored in the dark at four degrees.

Procedure with DAPI
During step 8 after incubation with the secondary antibody and two washes, include DAPI in the third wash (try a final concentration of 1:10,000 as DAPI is very bright)

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