8 In Situ Hybridization for RNA: Radioactive RNA Probe
119
Procedure
1. Write sample name, date, etc. on a small sheet of paper with
pencil.
2. Prepare sample vials for fixation and put the paper in it.
3. Prepare 50 ml fixative.
4. Prepare a syringe (5 ml), a needle (27G), two forceps, and two
pairs of scissors.
5. Anesthetize a mouse in a bottle containing a few ml of
ethylether.
6. Wait for 2 mins and dislocate mouse neck.
7. Perfuse the mouse gently through left ventricle after cutting
right atrium.
S. Remove tissues and cut the tissues in 3mm blocks.
9. Postfix the tissues overnight at 4°C in the fixative.
10. Wash with sterilized PBS three times for 15 mins to remove
fixative.
11. Dehydrate by passing the tissues through a graded series of
ethanol concentration for 15 mins: 50% on ice, 70%, 80%,
90%, 95%, (following processes may be performed at
room temperature.),lOO% three times, xylene three times,
xylene:paraffin 2:1 at 60° C for 1 h, paraffin for 1 h, and paraffin overnight.
12. Embed the tissues in paraffin at 62° C with the paper for
identification of sample.
13. Store blocks in refrigerator.
1. Put new slides in acetone containing 2% 3-aminopropyltriethoxy silane (APES) for 5 mins.
2. Wash the slides in acetone, water and ethanol for 5mins each.
3. Dry the slides at 37° C in a clean incubator.
1. Sterilize everything with autoclave or 70% ethanol.
2. Trim block surfaces.
Tissue fixation
and paraffin
embedding
Preparation of
slide glasses
Sectioning
119
Procedure
1. Write sample name, date, etc. on a small sheet of paper with
pencil.
2. Prepare sample vials for fixation and put the paper in it.
3. Prepare 50 ml fixative.
4. Prepare a syringe (5 ml), a needle (27G), two forceps, and two
pairs of scissors.
5. Anesthetize a mouse in a bottle containing a few ml of
ethylether.
6. Wait for 2 mins and dislocate mouse neck.
7. Perfuse the mouse gently through left ventricle after cutting
right atrium.
S. Remove tissues and cut the tissues in 3mm blocks.
9. Postfix the tissues overnight at 4°C in the fixative.
10. Wash with sterilized PBS three times for 15 mins to remove
fixative.
11. Dehydrate by passing the tissues through a graded series of
ethanol concentration for 15 mins: 50% on ice, 70%, 80%,
90%, 95%, (following processes may be performed at
room temperature.),lOO% three times, xylene three times,
xylene:paraffin 2:1 at 60° C for 1 h, paraffin for 1 h, and paraffin overnight.
12. Embed the tissues in paraffin at 62° C with the paper for
identification of sample.
13. Store blocks in refrigerator.
1. Put new slides in acetone containing 2% 3-aminopropyltriethoxy silane (APES) for 5 mins.
2. Wash the slides in acetone, water and ethanol for 5mins each.
3. Dry the slides at 37° C in a clean incubator.
1. Sterilize everything with autoclave or 70% ethanol.
2. Trim block surfaces.
Tissue fixation
and paraffin
embedding
Preparation of
slide glasses
Sectioning