5-1 植物組織印染法
在硝酸纖維紙上做組織印染
將新鮮的組織切口壓印在硝酸纖維紙上 20-30 秒，隨即將印染過的紙放入
80℃抽真空之烘箱中約兩小時後即可染色或在顯微鏡下觀察。在印染的時候，
對每個材料所施的壓力求取一致是很重要的。
2007/3/8 生物顯微實驗技術 植物組織印染法 上課前準備清單
1. 材料：西洋芹
2. 硝酸纖維紙
3. 80℃抽真空之烘箱
4. 蒸餾水
5. 封片膠
6. 光學顯微鏡*1
7. 解剖顯微鏡*1
5-2 In situ Hybridization
1. Tissue Fixation in FAA
A. FAA (formalin-aceticacid-alcohol) for 100 ml
90%EtOH 50 ml
glacial acetic acid 5 ml
formaldehyde (37%) 10 ml
ddH2O 35 ml
B. Dehydration using Histochemical processor and embedded in 62℃ melted
paprffin
70% (in DEPC ddH2O) 30 min
80% (in DEPC ddH2O) 30 min
95% 30 min
100% 30 min x 3
Hemo-D 2 hr
paraffin infiltration for several days 7 days
embedded and solidification
C. Cut the sections by new knife and put the sections directly onto the slide which
was placed on heat block with one drop of DEPC-treated ddH2O. Let the
section rehydeate and dry on the slide.
D. De-Paraffin: Use slides with 1 or 2 sections close together (this reduces the
amount of solutions needed).
Deparaffinization: 2x, 5m - Hemo De
2x, 5m - 100% EtOH
1x, 5m - 50% EtOH (in DEPC ddH2O)
air dry completely, circle sections with rubber cement to keep solutions on
sections and for sealing coverslip.
2. Probe preparation:
A. plasmid construction: insert the desired cDNA into pBluescript vector (SK-)
(because the promoters used are T3 and T7 promoters in pBluescript vector)
B. template DNA preparation:
a. linearization of template DNA by restriction enzyme digestion (in DEPC
H2O)
b. check the linearity of the DNA by agarose gel electrophoresis
c. Phenol/chloroform
- add more TE buffer to final volume to 400 ml
- add 200 ml of phenol and 200 ml chloroform, shake well
- spin max speed x 5 min
- take the aqueous phase (top fraction) into new microfuge tube
- add 400 ml chloroform and shake well and spin max spped x 5 min
- the the top fraction into new microfuge tube
- ppt the DNA: add 1/10 final volume of NaOAc
- for isoproponal: add 200 ml isoproponal and 60 ml NaOAc (1/10 of 600 ml)
- for 100%EtON: add 1000 ml EtOH and 140 ml NaOAc (1/10 of 1400 ml)
- for the EtOH, sit on ice for 10 min then spin max speed x 5 min
- for the isopropanol, spin at max speed x 5 min
- rinse the pellet with 70%EtOH and air dry
- respspend DNA in TE buffer or DEPC H2O (the DEPC H2O is better, because
the TE buffer contained the EDTA which may interfer the reaction of T3/T7
polymerases)
C. Denature the template DNA by boiling (100℃) for 5-10 min.
D. DIG (digoxigenin)-labeling by in vitro transcription reaction for both sense and
antisense probes (Kit from Roche BM Cat# 1175025 and T3 RNA polymerase
Cat# 1031163)
Standard labeling reaction (from the BM):
1. Add the following reagents to a sterile. Rnase-free microfuge tube (on ice) in the
following order:
- 1 mg purified templae or 4 ml control DNA (vial 3 or 4)
- 2 ml NTP labeling mixture (10x), (vial 7)
- 2 ml transcription buffer (10x), (vial8)
- 1 ml RNase inhibitor (vial10)
- if desired add H2O, RNase-free to a final volume of 18 ml
- 2 ml RNA polymerase (T3 or T7) (vial 13 or 12)
2. Mix gentle and centrifuge briefly. Incubate for 2 hr at 37℃.
3. If desired, add 2 ml Dnase I, Rnase-free (vial9). and incubate for 15 min at 37℃
to remove the DNA template.
4. With or without prior Dnase I treatment, add 2 ml 0.2 M EDTA (0.4 ml of 0.5 M
EDTA + 0.6 ml DEPC H2O) solution to microfuge tube.
5. Add 2.5 ml 4 M LiCl and 75 ml prechilled 100% ethanol. Mix well and leave for
at least 30 min at -70 蚓 or 2 hr at -20℃.
6. Centrifuge for 15 min at 12000×g and wash the pellet with 50 ml 70% cold
ethanol (v/v).
7. Dry briefly under vacumm amd dissolve in appropriate volume of water,
Rnase-free. If desired, add 1 ml Rnase inhibitor (vial10).
E. Quantitation of the both sense and antisense probes and check the specific
activities by checking the incorporation of DIG to both sense and antisense
probes.
F. Probe hydrolysis and check the final probe size by the gel electrophoresis. For
better pentration of the RNA probe into the cell, the length of the probe should
be smaller than 200 bp probe hydrolysis:
-add equal volume of probe and hydrolysis buffer* and incubate for X min at 60
℃ equation: 1.2-desired probe size (in kb) (original probe size)×1.2× desired
probe size (in kb) 10
e.g. the orignial probe length is 1.1 kb ?1.1 /10 = 0.11
for 150 bp: (1.2-0.15) / (0.11 x 1.2 x 0.15) =53 min.
for 100 bp: (1.2-0.1) / (0.11 x 1.2 x 0.1) =83 min.
- to stop, add equal volume of neutralization buffer**
- add 1 ml glycogen and 3 volume of cold 100% EtOH
- precipitate at -20℃ for at least 2 hrs
- centrifuge @ 12000 ×g 20 min at 4℃ and wash the pellet with 70%EtOH.
- check the probe size by the gel electrophoresis
* Carbonate hydrolysis buffer:
0.0672 g NaHCO3+0.01272 g Na2CO3 in 1 ml DEPC H2O
(0.672 g NaHCO3 +0.1272 g Na2CO3 in 10 ml DEPC H2O)
** Neutralization buffer:
66.7 ml 3 M NaOAc+10 ml acetic acid+923 ml DEPC H2O
(667 ml 3 M NaOAc+100 ml acetic acid+9230 ml DEPC H2O)
3. Tissue section treatment:
A. Proteinase K (1 mg/ml) treatment for 30 min at 37℃ in TE buffer.
TE buffer: 50 mM Tris, 5 mM EDTA in DEPC water, pH 7.4
- protease treatment increases accessibility by digesting protein around target
nucleic acids.
B. Wash to remove proteinase K:
TE buffer 2 x 5 min, RT
DEPC water 2 x 5 min, RT
C. blocking with 0.1 % PVP (soluble) for 20 min at RT (optional and useful for
wood tissue)
D. Washing with DEPC water 2 x 5 min at RT
4. Prehybridization at RT for 2 hr in Hybridization solution + 1 l Dextran Sulfate
Hybridization solution 960 l
DEPC-H2O
160
5 M NaCl
57.2
50x TE buffer
19.2
50x Denharts
19.2
Formamide
480
salmon sperm DNA (ssDNA) 38.4 (10 mg/ml)**
t RNA
24 (10 mg/ml)
** the ssDNA were broken by sonicator and denatured by boiling for 10 min and
quickly putting on ice to preserve the single strand DNA.
- prehybridization is to prevent background staining
5. Denature the probe (used 0.05 mg/ml) at 70℃×5 min
6. Hybridization at 42℃ for overnight (see also notes*)
Hybridization Buffer+probe (1 ng/l)+1 l Dextran sulfate
- may also preheat probe to 70 蚓, 5m in water bath to remove secondary
structures
7. Washes
4x SSC 30 min, RT
2x SSC 30 min, RT
1x SSC 30 min, RT
0.1x SSC 30 min, at Hybridization temperature (i.e. at 42℃) for high stringency
-posthybrization washes help remove non-specific hybrids. Probe may bind to
sequence which bears homology but not entirely homologous. Wash with various
stringencies, can vary formamide conc., salt conc., and/or temp.)
8. Detection of DIG by AP-conjugated antibodies (Sheep Anti-DIG-AP, Fab
fragments from Roche BM Cat# 1093274)
A. Wet slides with TBST (10 mM Tris, 500 mM NaCl, 0.3%Tween 20)
B. Block with TBST-BSA (1%) for 1 hr at RT
C. Incubate with Sheep Anti-DIG-AP in TBST-BSA 1:500 dilution for 2 hrs at
RT.
D. Wash with
TBST-BSA 2×10 min at RT
TBST
2×10min at RT
E. Wash with AP developing buffer (100 mM Tris-HCl, 10 mM NaCl, 50 mM
MgCl2, pH 9.5).
F. Incubate with AP substrate (from Roche BM ) 450 mg NBT+175 mg BCIP 4.5
ml of 100 mg/ml NBT (4-nitroblue tetrazolium chloride)+3.5 ml of 50 mg/ ml
BCIP (5-Bromo-4-chloro-3-indolyl-phosphate) in 1 ml AP buffer in the
darkness (critical requirement) until color development is acceptable.
G. Stop the reaction by adding Tris buffer (10 mM Tris-HCl, 1 mM EDTA, pH
8.0)
H. Dry the slide under darkness.
I. Observe the labeling under microscopy.
* Notes on hybridization; hybridization depends on ability of denatured DNA to
reanneal with complementary strands in environment just below their melting
point (Tm=emp at which 1/2 DNA is present in single strand form (denatured)).
Stability of DNA directly dependent on GC content, increase molar ratio of GC
pairs=increase melting point (Tm).
Renaturation of DNA and Tm influenced by: temperature, pH, concentration of
monovalent cations, presence of organic solvents.
pH: increase pH above 6.5-7.5 to produce more stringent hybridization
conditions
Monovalent cations (sodium ions): interact electrostaticly with nucleic acids (i.e.
phosphate groups). Increase salt conc.=decrease electrostatic repulsion between
the 2 strands & increase stability of hybridization
Decreased salt concentration affects Tm and renaturation rate.
Formamide: Organic solvent, reduces melting temp of DNA/DNA, DNA/RNA
duplexes so that hybrid can occur at temp below 65- 75℃ (normal temp for in
situ to work, but causes trouble with morphology) with 50%formamide in mix.
Dextran Sulfate: strongly hydrated, macromolecules have no access to waterincreases hybridization rates by apparently increasing probe concentration.
Other variables;
Probe length - increase probe length=increase renaturation rate
Probe conc. - increase probe concentration=increase reanneal rate
**Solution (All the solutions are preparewd by DEPC H2O
A. FAA (formalin-aceticacid-alcohol) for 100 ml
90 %EtOH 50 ml
glacial acetic acid 5 ml
formaldehyde (37%) 10 ml
ddH2O 35 ml
B. TE buffer: for 100 ml
50 mM Tris,
5 mM EDTA
DEPC H2O
pH 7.4
C. Hybridization solution + 1 l Dextran Sulfate 960 l
DEPC H2O 160
5M NaCl 57.2
50x TE buffer 19.2
50x Denharts 19.2
Formamide 480
salmon sperm DNA (ssDNA) 38.4 (10 mg/ml)
tRNA 24 (10 mg/ml)
D. 20x SSC
1L
500 ml
3 M NaCl 175.3 g 87.65 g
0.3 M Na citrate
88.2 g 44.10 g
E. TBST
1L
10 mM Tris 10 ml of 1 M Tris-HCl, pH7.4
500 mM NaCl
100 ml of 5 M NaCl
0.3% Tween 20 3 ml
DEPC H2O 887 ml
pH 7.4
F. AP developing buffer
50 ml
100mM Tris-HCl, pH 9.5 5 ml of 1 M Tris-HCl, pH 9.5
10mM NaCl 1 ml of 5 M NaCl
50mM MgCl2 0.25 ml of 1 M MgCl2
DEPC H2O add to 50 ml
filtreated through 0.22 mm filter
G. Stop solution: Tris buffer
10 mM Tris-HCl
1 mM EDTA
pH 8.0
DEPC H2O
H. 0.1%PVP
Dissolve 0.1 g PVP in 100 ml DEPC H2O
I. 1 M Tris-HCl. pH 7.4 in 1 L
Dissolve 121.14 g Tris in 1 L DEPC H2O and add conc. HCl > 75 ml
J. I. 1 M Tris-HCl. PH 8.0 in 1 L
Dissolve 121.14 g Tris in 1 L DEPC H2O and add conc. HCl ~ 75 ml
K. 0.5 M EDTA, pH 8.0 (500 ml)
add 93.05 g EDTA and some NaOH into 400 ml DEPC H2O then adjust the pH
to 8.0 by adding more NaOH
L. 5 M NaCl,
add 146.1 g NaCl in 500 ml DEPC H2O
M. 4 M LiCl
add 16.956 g LiCl into 100 ml DEPC H2O, then autocleave
N. 3 M NaOAc
add 24.61 g NaOAc into 100 ml DEPC H2O, then autocleave
資料來源：Guang-Yuh Jauh, Institutie of Botany, Academia Sinica, Nankang,
Taipei, Taiwan 11529 R.O.C.