(Spreads adapted from Luis's 'Modesti/Giroux', immunostaining from
KS buffer (make fresh each time, only because it's
easy and will be clean)
|– 1.2 M sorbitol (from 2% filtered stock)
– 2% KAc (from 20% filtered stock)
Spheroblasting buffer (make fresh)
|– KS buffer + 1/100th
volume 1 M DTT + 1/100th volume zymolyase (from 10mg/ml
MS buffer (make fresh each time, only because it's
easy and will clean)
Add 1mM PMSF just before use (from 250X stock).
|– 1.2 M sorbitol
– 0.1 M MOPS (from 0.5 M filtered stock)
– 1 mM EDTA (from 500 mM filtered stock)
– 0.5 mM MgCl2 (from 1 M filtered stock)
Fixative (make fresh in fume hood)
- Put 2 g paraformaldehyde in 50 ml dH2O and 200 ul 1M KOH.
- Shake in 50 ml Falcon ~1 h at 30° C until dissolved.
- Add 1 ml 0.5 M MOPS.
Filter sterilise and keep. Vary concentration to increase spreading.
(You have to increase it considerably, 5-10%, to increase spreading
so try to improve the physical spreading etc. instead.)
|– 1% Kodak photoflo (Hard to get — original protocol
used 1% NP-40, which gives similar results.)
Blocking buffer (make fresh each time)
|– 0.1 g dried milk powder
– 0.25 g BSA
– 5 ml PBS
Preparing the cells
Preparing the slides
- Take 1.5-3 ml sporulating culture and keep on ice.*
- Spin down in small centrifuge 1 min at 5000 rpm.
- Resuspend in 1 ml (ice-cold) KS buffer.**
- Spin down 2 min at 6000 rpm.
- Resuspend in 300 ul spheroblasting buffer.
- 20 min at 37° C.
- Check cell wall digestion.***
- Spin down spheroblasts 3 min at 4000 rpm.
- Gently resuspend in (ice-cold) MS buffer.
- Spin down 3 min at 4000 rpm.
- Gently resuspend in 60-100 ul MS buffer.****
Spreading (done in fume hood)
- Boil ~5 cm water in a large glass beaker in microwave.
- Add 1/100th volume of 1 M HCl (for 0.01 M).
- Set up over Bunsen burner flame and stand slides up inside beaker
(matt end up).
- Bring to the boil (may have to cover top).
- Boil for ~10 min.
- Use forceps to take out slides and rinse them in 100% ethanol.
- Lay out to dry — keep dust free!
- Pipette 20 ul gently resuspended spheroblasts onto centre of
- Prepare 3 full pipettes to carry out steps 3-5 as quickly as
- Add 80 ul fixative.
- Add 40 ul detergent in a swirling motion — can try waiting
1min here too.
- Add 80 ul fixative is a swirling motion.
- Use final pipette on its side to spread central area of slide
without touching slide.
- Remove bubbles.
- Dry flat in fume hood.*****
N.B. If you are not immunostaining, it is that you wash spread 30
min in PBS before proceeding to DAPI step. Also, the original protocol
said to rinse dried spreads in 0.2% Photoflo. This is not necessary,
but it may be if you are not immunostaining.
- Wash 10 min in PBS.
- Drain slide (just 2 s), add 200 ul blocking buffer to central
area of slide.
- Incubate 10 min at room temperature in humidity chamber.
- Drain slide, add 200 ul 1° antibody in blocking buffer.
- Add plastic square, incubate 1 h in humidity chamber.
- Remove plastic square, wash twice ~10 min in PBS.
- Repeat steps 4-6 for 2° antibody (incubate and wash in dark!).
- Drain slide, add ~2 ul DAPI/antifade to centre of slide.
- Add coverslip, cover slide with a tissue and press gently to
spread DAPI and dry.
- Paint edges of coverslip with nail varnish.
* You can get away with less but aim for 2 ml as you do seem to lose
cells and it’s easier at the last stage if there are more cells
— wait at this stage until the end of the time course.
** The original protocol has 3 washes in 1ml KS buffer but this is
unnecessary and loses more cells — sometimes wash in less volume
if cells are particularly sticky and don’t spin down well (seems
to be more of a problem at 5-7 h).
*** Mix a 2 ul sample (from the bottom of the Eppendorf) with 2 ul
2% SDS on a slide, add coverslip and check >95% cells are lysed
(usually just see a whole lot of cell debris swirling around —
make sure you mix properly or it’s difficult to tell). Usually
20 min is enough for me but do it for longer if necessary. The only
times the cells were not well digested was when MS buffer was used
instead of KS, in which case, had to start again!
**** You can leave it at this stage overnight on ice if you want!
***** Original protocol says 2 h but always seems to need longer —
do at least overnight, often over the weekend! Don’t worry if
they do not look dry after overnight drying as they have a 'wet-look'!
Also, sometimes they look crystalline, which may seem worrisome, but
there is not a noticable difference at the microscope stage. You can
store spreads long term no problem in slide boxes at -80° C.
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