Edinburgh Core Database Group

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Gaido Group Protocols

Part 3 of 3 (previous)

GUDMAP – SOP “Fluorescent ISH – Freedom Evo” –
Gaido Lab - GenePaint System
The Hamner Institutes for Health Sciences – CIIT at the Hamner

Last update:  March 19, 2007

For questions and comments, please contact:
gaido@thehamner.org  Kevin Gaido
Updated By:  Janan Hensley

Stock solutions

DEPC H2O  In fume hood, add 1mL DEPC to 1L of H2O.  Allow DEPC to dissolve for 4
hours or O/N with shaking at 4ºC.  Autoclave on liquid cycle, allow DEPC H2O to
equilibrate to RT before using.

10x PBS:  For 1L add the following solids components:

Na2HPO4 14.4 g
KH2PO4 2.4 g
KCl 2 g
NaCl 80 g

 

Add ultra-pure water to a volume of 800mL and stir until solid components dissolve. Adjust pH to 7.5, add ultra-pure water to a final volume of 1000mL. Autoclave and store in 1L bottle at RT .

1X PBS DEPC:  Add 1 package of Sigma’s powdered PBS (P3813-10Pak) to 1L of H2O,
dissolve by stirring.  Proceed with directions for DEPC H2O as stated above.

20% (5X) PFA:  1L  (Note: PFA is extremely hazardous). 
Add 1mL 2N NaOH to approximately 500 mL autoclaved water and heat the water to 65º C.  Under the hood, dissolve 200 g paraformaldehyde in the water while heating and stirring.  Add 100 mL 10x PBS and let the solution cool to room temperature.  Adjust pH to 7.4 using approximately 1 mL 2M HCl.  Adjust the volume to 1 L with H2O.  Aliquot out to 50mL or 10mL conicals.  Store at -20º C for up to 6 months, or at 4º C for up to one week.  Dilute to 4% (1x) for use. pH: 7.4.

4% PFA in PBS:  40mL of 5X PFA added to 160mL PBS DEPC H2O.  (acetylation)

Acetylation Buffer:  200mL DEPC H2O, 2.66mL Triethanolamine, 350µl 37% HCl. 750µl (x2) is added when slides are incubating.  (acetylation)

70% EtOH:  Make 200mL each container for acetylation.  Mix 140mL EtOH plus 60mL
DEPC H2O.  Discard after 2 weeks.

80% EtOH:  Make 200mL each container for aceylation.  Mix 160mL EtOH plus 40mL
DEPC H2O.  Discard after 2 weeks.

95% EtOH:  Make 200mL each container for acetylation.  Mix 190mL EtOH plus 10mL 
DEPC H2O.  Discard after 2 weeks.

100% EtOH:  Make 200mL.  Discard after 2 weeks.

20x SSC:  Pre-made: Ambion cat# 9763 (1L)

OR for 4L add the following solid components:

Citric acid 325.8g
NaCl   701.2g

 

Add ultra pure water to a volume of 3600 mL and stir until solid components dissolve.
Adjust pH to 7.0, add H2O to a final volume of 4000mL.  Autoclave and store in
1L bottles at room temperature. 

5x NTE: For 1L add the following solids components:

NaCl     146.25g
Tris    6.05 g
EDTA    9.3 g

 

Add ultra-pure water to a volume of 800mL and stir until solid components dissolve. Adjust pH to 8 (fine tune the pH), and add ultra-pure water to a final volume of 1000mL. Autoclave and store in 1L bottle at RT .

10x TNT: For 1L add the following solids components:

Tris (Trizma base)  121.5 g
NaCl       87.95 g

 

Add ultra-pure water to a volume of 800mL and stir until solid components dissolve. Adjust pH to 7.5, add ultra-pure water to a final volume of 1000mL. Autoclave and store in 1L bottle at RT .

Sheep serum:
Heat the inactive sheep serum for 30 minutes at 55º C on a shaking water bath.   
Allow the solution to cool and aliquot.  Store at -20º C.

TNB: Add 0.25g of Perkin Elmer blocking powder to 50mL 1xTNT.
                       
Heat it in 65ºC waterbath for about 1 hour. Needs to be degassed. Make sure it has cooled down to RT before adding primary antibody (do not cool rapidly or with ice).

Maleate Wash Buffer (1X MWB):  For 1L add the following solids components:

NaCl    8.765 g
Maleic Acid  11.61 g
NaOH              8 g

 

Dissolve in 800m L ultra-pure water and adjust pH to 7.5.  Add ultra-pure water to 1000mL.  Store at 4°C.  Discard after 1 week.

Blocking Reagent 1%: For 50mL add the following solids components:

0.5g of Roche blocking powder and fill up to 50mL with MWB.
                       
Heat it in 65ºC waterbath for about 1 hour. Need to be filtered and degassed.  Before adding secondary antibody make sure it is at RT.

TMN:

Tris (Trizma Base)(0.1M) 12.125 g
NaCl (0.1M) 5.85 g


Dissolve in 800mL of ultra-pure water,

Adjust the pH to 9.5.
Add 10.11g MgCl2(6H2O) and readjust the pH to 9.5. Add ultra-pure water to 1 L.


Appendix: List of chemicals

Chemical

Catalog #

Amount

DEPC

Sigma D5758

50mL

PBS

Sigma P3813

10 pack (makes 10 – 1 Liters)

2M HCl

Fisher A144-212

2.5L

Tween 20

Fisher BP337

500mL

20X SSC

Ambion 9763 (ABI )

1L

Formamide *

Roche 11814320001

500 mL

Iodoacetamide **

Sigma I-1149

100 g

Sheep (lamb) serum  *

Gibco 16070-096 or (Sigma S-2263 (100mL))

500 mL

Roche Blocking reagent

Roche 11096176001

50g

Levamisole *

Sigma L9756

10 g

tRNA (yeast)

Ambion 7119

0.5mL

heparin

Sigma H-3149

100K units (grade 1A from Porcine intestinal mucosa)

In situ Hybridization buffer *

Ambion B8807G

100 mL

Hydrogen peroxide (H2O2) *

Sigma 216763

500 mL

Methanol (MeOH) *

Fisher A412-4

4 L

TSA-Plus Cy3 (Cyanine 3 Tyramide) *

Perkin Elmer
NEL744

50-150 slides

1X Plus amplification diluent *

Perkin Elmer NEL744

15mL (included in TSA-Plus Cy3 kit)

Paraformaldehyde * *

EMS 19208

0.5 kg

Triethanolamine

Fluka Biochemika 90279

500mL

HCl (concentrated)

Sigma 258148-500mL

500mL

Acetic anhydride

Sigma A6404-200mL

200mL

Anti-digoxigenin-POD *

Roche 11207733910

1 mL

Potassium chloride (KCl) *

Sigma P4504

500 g

Sodium phosphate (Na2HPO4) *

Sigma S0876

500 g

Potassium phosphate (KH2PO4) *

Sigma P5379

100 g

Sodium citrate (citric acid) *

Sigma C7254

1 kg

Tris (Trizma) *

Sigma T1503

1 kg

Blocking reagent [TNB] *

NEN FP1012

10 g

Maleic acid *

Sigma M0375

500 g

Magnesium chloride (MgCl2*6H2O) *

Sigma M0250

500 g

Sodium hydroxide (NaOH) *

Sigma S5881

1 kg

EDTA *

Sigma E4884

500 g

Diethylpyrocarbonate (DMSO)*

Sigma D8418

50mL

Sodium Chloride (NaCl)

Sigma S3014

500g

DTT – DL-Dithiothreitol

Sigma 43815

1g

OTHER SUPPLIES

 

 

Superfrost Plus slides

Fisher 12-550-15

 

Corning cover glass No. 1

Corning 2935-224

10pks/case

VectaShield Mounting medium for Fluorescence with DAPI

H-1200

10mL

*Handle all chemicals with care.

 

 

Solution descriptions

In situ Hybridization buffer (Ambion B8807G, ABI)
SSC (0.3M NaCl 0.03M sodium citrate trisodium citrate dehydrate pH 7.0).
NTE (5mM EDTA,  10mM Tris, 500mM NaCl, pH 7.6)
TNT solution (100mM Tris 150mM NaCl, pH 7.6)
TNB blocking buffer (0.5% blocking reagent in TN (PerkinElmer Lifesciences, FP1020) pH 7.6).
MWB (100mM maleate, 150mM NaCl, pH 7.5)
Blocking reagent 1% (1% BR) (Roche 110961760001) containing MWB
TMN solution (0.1M Tris, 0.05M MgCl2, 0.1M NaCl, pH 9.5)

Summary of Tecan script


cycles

T (min)

volume

Reagent

l. class

temperature

5

5 min.

300

H2O2 in MeOH

ethanol 

24º C

7

5 min.

300

PBS (1)

water

24º C

2

5 min.

300

0.2M HCl

water

24º C

7

5 min.

300

PBS (3)

water

24º C

2

15min

300

hyb-mix (1)

hyb-mix

24º C

1

15 min.

 

Heat up to 64º C

hyb-mix

64º C

1

330 min

300

probe hybridization

probe

64º C

5

5 min.

300

5 x SSC

water o/n

62º C

5

10 min.

350

formamide I

water o/n

62º C

5

12 min.

350

formamide II

water o/n

62º C

3

8 min.

300

0.1 x SSC (1)

water o/n

62º C

1

8 min.

300

0.1 x SSC (2)

water o/n

24º C

4

5 min.

300

NTE (1)

water o/n

24º C

6

5 min.

300

20 mM iodoacetamide (1)

water o/n

24º C

4

5 min.

300

NTE (2)

water o/n

24º C

2

5 min.

300

TNT (1)

water o/n

24º C

6

5 min

300

4% sheep serum (1)

water o/n

24º C

4

5 min.

200

TNT (2)

water o/n

24º C

2

10 min.

300

TNB blocking buffer

water o/n

24º C

2

5 min.

200

TNT (3)

water o/n

24º C

2

5 min.

300

maleate wash buffer (1)

water o/n

24º C

2

10 min.

350

blocking reagent

water o/n

24º C

2

5 min.

300

maleate wash buffer (2)

water o/n

24º C

2

5 min.

250

TNT (4)

water o/n

24º C

3

5 min.

350

TMN

water o/n

24º C

4

5 min.

200

TNT (2)

water o/n

24º C

4

10 min.

300

TNB blocking buffer

water o/n

24º C

2

30 min

350

antiDIG

water o/n

24º C

6

5 min.

200

TNT (3)

water o/n

24º C

1

25 min

250

TSA-Cy3

-

24º C

4

5 min.

250

TNT (4)

water

24º C

2

5 min.

400

TMN

water

24º C

3

x

400

System liquid (1)

System liquid

24º C

1

x

300

NTE (3)

water

24º C

1

10 min.

200

4% PFA (2)

water

24º C

3

x

400

System liquid (2)

System liquid

24º C

Platform layout

Left front                                                         Left rear


PreHyb

HCl

MeOH/PFA

TMN

 

Serum

NTE

TNB

TNT (extra)

MWB

BR

IDO

PBS

TNT

 

Anti-DIG

TSA

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5 x SSC

 

Formamide I

 

0.1 x SSC

 

 

Formamide II

Right Front                                                   Right rear


Group Description