Hank Bass' FISH page (yeah, I know ...)

EMBEDDING CELLS
revised 12/99, hwb, version4 

MBA = Complete Meiocyte Buffer A (1xBFA salts, 1X DTT, 1X PA, 0.32 M sorbitol

1. Make 3X = 15% Acrylamide (fr. 30% stock [33:0.3], st. filt'd, stored 4 C.)
    ----------------------per 10 mL (ul = microLiter)---------
       5 mL   30% Acryl
       1 mL   10X BufferA - - - , (salts, pH'd)
      10 uL   DTT, 1000X
      10 uL   PA, 1000X
       2 mL   1.6M Sorbitol (st. filt'd, stored -20) - to 0.32 M final
       2 mL   dei H2O, to 10 mL
    ----------------------------------------------------------
2.  Aliquot into 1.5 mL tubes, 0.5 mL per tube, degas 1 min (speedvac).
3.  Make 1 mL 20 % sodium sulfite, anhydrous.
4.  Make 1 mL 20 % APS.
5.  Microdissect fixed tissue into MBA.
6.  Add 12 uL cells in MBA to slide with nail polish ring. 
7.  Add 6 uL activated acryl mix, swirl to mix.
      activated acryl mix = 
        500 uL 3X acryl mix + 25 uL sulfite, + 25 uL APS (you have 15 seconds).
8.  Cover with coverslip for 20 min.

HYBRIDIZATION

1.  Remove coverslip & wash acryl pad with MBA, 4X.
2.  Add prehyb (50% formamide, 2X SSC), 4X.
3.  Add probe/hyb (50% formamide, 2X SSC, probe), 2X
4.  After second addition of probe, add coverslip & seal with rubber cement.
5.  Place on 35-40 C slide warmer 15-20 min.
6.  Denature 96 C, 6 min  on PCR block
    (optimal temp and time to be determined with a temp series expt.)
7.  Hyb overnight, 37 C

WASH, DAPI, and MOUNT (use PBS, not TBS)
    Each WASH is 100-200 uL, on rotary shaker, 5-10 min, RT, dark

1.  Remove coverslip.
2.  Do the washes, 1 mL per slide
    WASH-1.  3 times (4xSSC)
    WASH-2.  3 times (2X SSC, 2X PBS)
    WASH-3   3 times (1X PBS, 0.2% tween-20)
    WASH-4.  3 times (1X PBS, 1mM (=1X) DTT)
    WASH-5.  3 times (1X PBS)
    WASH-6.  2 times (1X PBS, 10 ug/mL DAPI), 2nd wash for 20 min, RT.
    WASH-7.  3 times (1X PBS, 1mM DTT (=1X) DTT)
3.  Clean slides thoroughly and mount in VectaShield (Vector Labs)
    Add and remove vectashield 2X, the third time, boink mount 
    with 20 uL on cs (22x22) or 30 uL on cs (22x30).
4.  Seal w/ Sally Hansons Clear Hard as nails, dry to completion
5.  Achieve scopage or store -80 in dark conical (2 per, back to back)
------------------------------------------------------------------------

The reference is:
Bass H.W., Marshall, W.F., Sedat J.W., Agard D.A., and Cande W.Z. (1997)
 Telomeres cluster de novo before the initiation of synapsis; a 3-dimensional
 spatial analysis of telomere positions before and during meiotic prophase
J Cell Biol 137(1):5-18

The longer detailed version is here:
FISH_meth.html