酵母免疫熒光染色--Immunofluorescent Staining of Yeast:SDS Permeabilization Metho
Materials:
1) 1.0M KPO4 pH 6.5 : Make by mixing 33 mls of 1M K2HPO4 with
67 mls of 1M KH2PO4
2) 0.1M KPO4 pH 6.5
3) 0.1M KPO4 pH 7.5: Make 500 mls by mixing 41.7 mls of 1M
K2HPO4 and 8.3 mls of 1M KH2PO4 with 450 mls of ddH2O
4) 37% Formaldehyde
5) KS Buffer: 0.1 M KPO4 pH 7.5/1.2 M Sorbitol
6) Zymolyase 100T: 5 mg/ml solution in 0.1M KPO4/0.5% β-
mercaptoethanol
7) β-mercaptoethanol
8) HS Buffer: 0.1 M Hepes pH 7.4/1.0 M Sorbitol
9) HS/SDS Buffer: 0.1 M Hepes pH 7.4/1.0 M Sorbitol/0.5% SDS
10) 0.1 % Polylysine (MW >300,000; Sigma Cat. No. P-1524) Stored
in 1ml or 10 ml aliquots at -20°C. After thawing microfuge
aliquot for 5 min. at 4°C before adding to slides.
11) Multiwell teflon masked slides (Carlson Scientific Inc. Peotone
IL. Cat. No.100806)
12) PBT Buffer: PBS with1 mg/ml BSA and 0.02% Tween 20
13) PrimaryAntibodies: Affinity purified polyclonal antibodies or
monoclonal antibodies from cell culture supernatant (notfrom
ascites fluid). Dilutions must be determined emperically for each
primary but common ranges are for affinity purified sera 1:10
to 1:100 in PBT Buffer. For cell culture supernatants 1:1 to 1:5
dilutions are normally used (if cell supernatants were
concentrated by ammonium sulfate precipitation then 1:10 to1:50
dilutions are often used). After diluting in PBT buffer spin
secondary in microfuge (at 4°C) for 5 min to pellet any debris.
14) SecondaryAntibodies: Jackson Immunoresearch Laboratories
makes high quality secondary antibodies which are good for
both single and double labeling experiments. We've been using:
Texas Red conjugated AffiniPure Donkey Anti-Mouse IgG
(Cat#715-075-141) and Fluorescein (DTAF) conjugated
AffiniPure Donkey Anti-Rabbit IgG (Cat#711-015-132). They
are sent as freeze-dried powder which should be reconstituted
with ddH2O according to directions and then aliquoted into 50
μl/tube (marked with name of Ab and Date) and frozen on dry
ice and stored in the "Secondary Antibodies" Box in the -80°C
Freezer. After thawing an aliquot keep it at 4°C--it should be
good for about a month. These secondary Abs work very well
for both double and single labeling and should be used at 1:50
to 1:100 dilutions each (in PBT buffer). After diluting in PBT
the solution should be microfuged for 5 min at 4°C to remove
any precipitates that may have formed.
15) Mounting Medium: Dissolve 10 mg of ρ-phenylenediamine
(Sigma Cat. No. P-6001) in 1 ml of 1M K2HP04 in a 1.5 ml
eppendorf tube. Vortex vigorously for several minutes, cover
with foil, and rock on nutator for 20-30 minutes. Vortex again
and microfuge for 5 min. Remove the top 800 μl to a 15 ml
tube. Add 7.2 ml of glycerol and mix by vortexing. Divide into
500 μl aliquots and store at -80°C (in the "Secondary Ab" Box).
Optional: For nuclear staining DAPI can be added to mounting
medium as follows: make a 1 mg/ml DAPI (4',6-diamidino-2-
phenylindole dihydrochloride) solution in water, dilute it 1:100
with water and add 2.25 μl to 1 ml of mounting media.
Method:
1) For each strain to be examined inoculate 50 mls of YPD or SD
(with the appropriate amino acid supplements). Grow overnight at
25°C until culture is at a OD599 between 0.5 and 0.8. For examing
unshifted cells proceed directly to Step 2. For examining sec7ts and
sec6ts strains spin cells out of YPD at OD599 of 0.5-0.6 and resuspend
in the same volume of YP with 0.2% Glucose and then incubate in a
37°C shaking water bath for 2 hrs. Check OD599 and then cool for 5
min on ice until the culture is at room temperature before fixing.
2 ) Fix yeast directly in the culture by adding to each 50 mls of
culture: 5 mls of 1.0 M KPO4 pH 6.5 and 6 mls of 37% Formaldehyde
directly to the culture flask. Incubate at room temperature with
gentle shaking for 30 min. Transfer an amount of cells that is equal
to about 5-10 OD599 units for each culture into a 50 ml centrifuge
tube and spin in the Beckman table-top centrifuge for 5 min. at 2000
RPM at 25°C. Aspirate off the supernatant and resuspend each pellet
in 5 mls of 0.1 M KPO4 pH 6.5 and transfer to a 15 ml centrifuge
tube. Add 0.6 mls of formaldehyde to each tube and incubate at
room temperature for 1.5 hrs with gentle rocking.
3) Harvest fixed cells by centrifuging at 2.2K RPM for 5 min. at
25°C. Aspirate off supernatant and resuspend in 5 mls of 0.1 M KPO4
pH 7.5 and repeat spin. Aspirate off sup. and resuspend cells in 5
mls of 0.1 M KPO4/1.2 M sorbitol. Fixed cells can now be stored
overnight (or for several days) at 4°C.
4 ) Prepare Zymolyase solution by dissolving 2-5 mg of Zymolyase
in 0.1 M KPO4 pH 7.5 to give 5 mg/ml solution. Add β-
mercaptoethanol to 0.5 % (i.e. 5 μl/ml), mix by gently vortexing and
let sit at room temp. for 20-30 min to dissolve.
5) Harvest fixed cells at 2.2K RPM for 5 min. Aspirate off
supernatant and resuspend in 1 ml of 0.1 M KPO4 pH 7.5/1.2 M
Sorbitol. Transfer cells to a 13x100 mm glass culture tube (i.e. blue
capped tubes). Add 45 μl of 5 mg/ml Zymolyase solution and 5 μl of
β-mercaptoethanol and incubate at 30°C for 30 min with occassional
mixing by finger-flicking.
6) Harvest spheroplasts at 2.2K RPM for 5 min at 25°C. Resuspend
cells in 3 mls of HS Buffer and spin at 2.2K RPM. Aspirate off
supernatant and repeat wash one time.
7 ) To permeabilize cells resuspend spheroplasts in 3 mls of
HS/SDS and incubate at room temperature for 5 min. Centrifuge at
2.2K RPM for 5 min and resuspend cells in 3 mls of HS Buffer and
spin at 2.2K RPM. Aspirate supernatant and repeat wash with HS
Buffer. Resuspend cells in 1 ml of HS Buffer. Do not store the cells
for more than a few hours at this stage.
8 ) Set up an aspirator with "pulled" pasteur pipette at the end
near to where the slides are to be processed to speed up the washing
procedures. Prepare slides by adding 20 μl of 0.1% polylysine to
each well and incubate for 5-10 min. Aspirate off and wash each well
3 times with one drop of ddH2O.
9) Place 20 μl of spheroplasted/permeabilized cell suspension on
each well. Incubate for 5-10 min. Aspirate each well and add one
drop per well of PBT Buffer. Repeat twice and let sit while primary
Ab dilutions are prepared.
10) Prepare dilutions of primary antibodies in PBT and microfuge
before use. Add 20 μl of diluted primary antibody and transfer slides
to a plastic box with a moistened paper towel at the bottom.
Incubate 60-90 min.
11) Carefully remove slides from box to benchtop for washing. To
wash slides rapidly, hold aspirating pipette in one hand and another
pastuer pipette with a rubber bulb containing PBT Buffer in the
other hand. For each well aspirate the liquid and then add one drop
of PBT, aspirate add PBT, repeat this twice (leaving the well with a
drop of PBT) and then move on to the next well. It is important that
the wells do not dry out once antibodies have been added. Once all
the wells have been washed move on the next slide. Then repeat
this entire procedure twice so that each well has been washed at
least nine-times. This may seem excessive but it often dramatically
increases the quality of the images obtained and once you get the
hang of using both hands it can be done fairly quickly.
12) Wrap plastic box used for primary Ab incubations in foil so it is
light tight. Place slides inside and place 20 μl of secondary antibody
in each well. Incubate for 60-90 min at room temperature.
13) Wash the wells as before with a total of at least 9-10 washes of
PBT Buffer. Aspirate the wells dry after the last wash and let air dry
underneath a piece of foil (make a tent) for 10-15 min.
14) Place 4 evenly spaced small drops of mounting medium in
between the two columns of wells in the middle of the slide.
Carefully place the coverslip over the slide and let mounting medium
spread. Seal the slides with nailpolish, let dry 10-15 min. Then rinse
off slides with ddH20, dry with a kimwipe and view immediately or
store in dark at -20°C.
For More information and alternative methods see:
"Immunoflourescence Methods for Yeast" by Pringle et.al on pg. 565
in GuidetoYeastGenetics andMolecularBiology edited by Guthrie
and Fink (Vol. 194 of Methods in Enzymology).
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