5x Buffer PB (= 250 mM phosphate buffer, pH 7.2 after dilution)
To make 500ml,
Mix 140ml 0.25M NaH2P04
+ 360ml 0.25M NaH2P04
1x PB, 500ml
35 mL of 0.2 M NaH2P04
90 mL of 0.2 M NaH2P04
1x PB required for the procedure below
– Weigh 2g Triton on balance by pipeting into the tube,
– Make up to 10 ml with distilled water
– Stone overnight
4% paraformaldehyde (STIR IN chimney)
To make 50 mL,
– the use of ~ 40 mL of water to dissolve 2g paraformaidehyde
– add 200ul 0.2N NaOH and heat, while stirring in the chimney
– once dissolved, + 10 mL 5x buffer PB
– freezing ~ 10-15mL aliquots at 20 freezer.
Normal Goat SerumNGS
– store in aliquots in freezer
– if after repair cells, leaving them wet in the fridge, covered with a PB of washing.
– Be careful when pipeting and removing the solution; if not, the cells will come from.
Immunocytochemistry and Immunofluorescence Staining Protocol
1. Wash 1 x with PB
2. Fix with 4% paraformaldehyde (10 min., 15-30niin. If the cells easily come from)
3. Wash 3x with PB (5 min. Each)
4. preincubation (. 15 minutes) for each 1 mL PB + 1 00uL NGS, 10ul of 20% Triton
5. Primary antibody: for each total volume 1mL, add 20uL NGS, 5uL triton (2hrs); primary dilution varies with each antibody and must be tested empirically or in accordance with the direction of the manufacturer
6. Wash 3x with PB (10 min. Each)
7. Secondary antibody: selecting the correct (either goat anti-mouse or goat anti-rabbit) Cy3 (1/800 dilution): for each 1 mL PB, secondary 1.25uL, 5uL triton, 25uL NGS (30 minutes-don ‘ t go too long, or else high background)
8. Wash 3x with PB (15 min. Each)
9. Remove mowial for disbursement; take insurance slip, drain bit with Kleenex, + 20uL mowial on cells and covered with the cover glass
10. Turn the cover slip on a slide
11. nail polish place in the four corners of the cover glass. When the nail polish dries, put nail polish around the slide of the cover glass.
12. You can see it or put it in the dark at four degrees. Slides can be stored in the dark at four degrees.