FIXING AND IMMUNOFLOURESCENCE (EUKARYOTIC…) BETH 3/7/02
NOTE: THIS PROTOCOL USES MeOH FIXATION, SEE OTHER FIXING PROTOCOLS FOR CHEMICAL FIXATION
Preparation: PBS solutions in squeeze bottles, water bath at 37°C, 15 minute spin down and dilution of antibodies, labeling of slides. Get 35mm Tissue culture dishes and place Whatman paper (Fisher Brand P5 cat no 09-801BB, Diam 3.5cm) into the cover of the dishes and moisten with water or PBS.
1) Place copeland jars or 35mm tissue culture dishes filled with 100% MeOH containing 1mM EGTA into the –20°C. Transfer the slide from the warm dish (must be kept warm until right before the cold MeOH to preserve MTs). Dab off any excess media before transfer. Fix cells at –20°C for 5-10 minutes and place back into a dish that contains 1X PBS (pH 7.4).
2) Rinse one time in 1X PBS, to do this use vacuum atttachment and suck out the PBS from the dish, then using the squirt bottle, squirt new PBS onto the side of the dish, do not squirt onto the slide or you will dislodge cells. The reason that you suck off the liquid rather than lifting the slide into or out of the liquid is that any dust particles etc tend to get on the slide when you lift it up.
3) Block the slides one hour in 1X PBS + 1% BSA + 0.01% azide (pH 7.4) OR the PREFERRED Blocking is in 5% Goat serum + 1% BSA + 0.01% azide. Place all the dishes in fridge in a box lined with wet paper towels and covered with parafilm.
4) About 15minutes prior to the end of blocking, spin down the antibodies or dyes in the 4°C centrifuge at high speed to get rid of junk and dust. Make up the correct dilutions in 5% goat serum/1% BSA blocking solution.
5) After 1 hour, suck off the blocking solution and rinse the slides in 1X PBS + 1% BSA. Remove coverslip and PBS from dish, add a filter paper to the bottom part of the dish, wet the paper with a drop or two of water and replace slide being careful not to invert the slide and lose track of which side the cells are on. These filters will keep the slide from drying out.
6) Place 20-25 micorliters of the correct primary antibody dilution s onto the center of the coverslip. A good example to start with for determing primary concentration to use is (straight, 1/4, 1/10, 1/50, 1/100).
7) Incubate at room temperature or 37°C in the water bath on a pedestal to keep the slides from drying out. Incubate 1-2 hours or O/N at 4°C.
8) Remove coverslip and lean on the side of the dish, remove filter paper, replace slide and wash the slide 1X PBS 4 times for 5 minutes each, then 1X in PBS + 1% BSA, this final wash aids in making the coverslip wettable.
9) Repeat steps 5-7 for the secondary antibody. Incubate for 1 hour at RT for most secondaries. After a half hour start spinning the Mowial + 1/2% N-propyl gallate to rid of junk etc.
10) After incubation with the secondary, remove coverslip and lean on the side of the dish, remove filter paper, replace slide and wash the slide 1X PBS 4 times for 5 minutes each, then wash quickly 2X in H2O. Only do the H2O step one slide at a time and directly mount the coverslip onto a slide with a blob of 25microliters of Mowial /2% N-propyl gallate. Suck up any excess oozing with the vacuum tip. Leave O/N at 4°C before looking at OR sealing with nail polish. If the slides are blurry the next day you sealed them too soon.
NOTES: Sigma Immunochemicals sells Goat Serum and we have some in our tissue culture box in the –20 freezer. Product no. G-9023.
Mowial is a polymer like PEG, it is a water miscible mounting media. N-propyl gallate is an antifade solution, I think that both these can be bought as powders.
(Methods in Cell Biol, V 24 pp 113) Calbiochem corp 475904
while mounting medium is designed for maximum fluoresence emission of FITC, it is not intended to minimize fading of fluorescence over time.
a) Put 6g of glycerol in 50mL tube
b) Add 2.4g MOWIAL 4-88 into glycerol and stir thoroughly.
c) Add 6mL dH2O and leave solution at room temp for 2 hours (do not skimp on this!)
d) Add 12mL 0.2M Tris-HCl, pH 8.5 (2.42g/100mL).
e) Add n-propyl gallate (final concentration should be 0.5% mass/volume). Initial brightness decreases as more NPG is added, although with higher concetration of NPG, fluorescence bleaches less.*
f) Incubate in water bath at 50°C for 10 minutes with stirring to dissolve MOWIAL.
g) Centrifuge 5000g for 15 minutes
h) Aliquot and store –20°C.
The original recipe is 4% however this interferes with the optics of low signals. Though, when bleaching is high I have increased it to 2% +1% NPG.