FISH steps.

1, dewax:
1) xylene dewax 3 times, 5min each time;
2) 100% alcohol twice, 2min each time
3) remove alcohol, diagonal
slice
, mark the last section down, air
dry
.2,
protease
treatment:
1) 40 ml protease K digestive solution per dyeing cylinder, preparation method is as follows× 2×SSC 40 ml pouring Facal tube, warm up in the bath. Add the digestive enzyme fluid to the tube and shake until the enzyme dissolves.
2) Preheat the dyeing cylinder and protease K solution in a 37-degree C bath. Incubate 20min at 37 degrees C.
3) × SSC rinses the slices at room temperature 3 times, 1min at a time.
4) gradient alcohol dehydration (-20 degrees C pre-cooling).3, denaturation:
1) each vertical dyeing cylinder preparation 40 ml denaturation solution;
2) 78 degrees C bath balance preheat mixture dye cylinder;
3) Incubate 8min at 78C;
4) i.e. move 2min into a dyeing cylinder with -20C pre-cooled 70% alcohol, then move into 80%, 90% and 100% -20C pre-cooled alcohol in turn, drying air in 2min;
5) per cylinder.4, hybridization:
1) prepare the probe;
2) take a larger wet box, cross-position the slice;
3) drop 10 sl probe on the slice's
tissue
, cover the slide;
4) cover the wet box cover, 37 degrees C incubation 12h to 16h.
after hybridization:
5) tweezers carefully remove the cover slide;
6) 43 degrees C preheats the 40 ml washed slice 15min;
7) 2×SSC (37c) washes twice, 10min each time;
8) slices release the human dyeing cylinder within 1×PBS for testing, do not make the slice dry.
:
9) Remove slices from 1×PBS, remove excess moisture, and avoid dry specimens. Place the slices in a wet box and process 4 slices at the same time.
10) Each slice uses 30 μl to 60 μl rodin anti-digoxin
antibody
or FITC lye, incubate 20min at room temperature;
11) to remove the plastic cap film and place the slice in a dyeing cylinder containing 1×PBS. 1× wash 3 times at PBS room temperature, 2min at a time.
Amplification:
12) remove the slice from 1×PBS, diagonal slice to drain the liquid;
13) each slice drops 30 μl to 60 μl anti-ovopyanin antibody, plus plastic cap film, room temperature incubation 20min;
14) remove the plastic cap film, put the slice into a person with 1×PBS staining cylinder. 1× wash 3 times at PBS room temperature, 2min at a time;
15) remove the slice from 1×PBS, oblique slice to drain the liquid;
16) each slice drops 30 μl to 60 μl anti-lybin antibody, plus plastic cover film, room temperature incubation 20min;
17) 1×BBS room temperature wash 3 times, 2min each time.5, cell nucleation staining:
1) sheet slice plus 10 μl to 20 sl DAPI, cover the cover glass and incubate 2 to 5 ml at room temperature;
2) as soon as possible in the fluorescent
microscope
observation or closed box stored in the -20 O refrigerator. Slices can be observed under a microscope within 1h after dyeing..