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 (MgCl₂6H₂O)	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