Sterilizations

Bake all the items mentioned below at 180 deg for over night.

1. Glass cylinders –Different sizes – 1000ml – 2, 500ml, 100ml, 50ml
2. Glass bottles (remove the orange cap and the plastic ring and soak it in the 0.1N NaOH)
3. Spatulas – different sizes
4. Stir bars-very small sizes for xylenes. Different sizes.
5. Razer blades.

Keep all the glass items in the Big Oven and turned to Heat to 180 deg.

NaOH washing for plastic Tubwares.

Make 0.1M NaOH solution

NaOH—-4Grams/Liter. Soak all the Plastic Containers in NaOH water for overnight, cover it. Next day wash well with 17.5 megaohm water.

DEPC Treated water.0.8ml of DEPC/Liter

(to make RNAse and DNAse free water)

Make 6 Flasks of 2500ml of water each. DEPC treatment should be done in the Hood.

Add 2.0 ml of DEPC to 2500ml of 17.5 megaohms water. Cover it with parafilm and shake well until DEPC dissolves in water. Then cover with Aluminium foil loosely and shake to wet the rim of the flask and leave it in the hood for Overnight. Next day Autoclave for 30 minutes at 121 degree celsius.

Insitu pre- Hybridization

6X Salts

3.6 mL — 5M NaCl (DEPC treated)

0.6 mL — Tris 1M pH 6.5 (use DEPC water but do not mix DEPC in Tris Buffer)

3.0 mL — 0.2M NaPO4 pH 6.8(DEPC treated)

0.6mL — 0.5M EDTA (DEPC treated)

2.2 mL — DEPC water

———–

10 mL —- Total

Pre – Hybridization Mix for 100 slides ( without formamide)

6X salts —–                           3333.00 uL

Dextran Sulfate(50%)——    4000.00 uL

50X Denhards —————-     400.00 uL

t-RNA (100mg/mL)———— 200.00 uL

DEPC water —————–       567.00 uL

8500.00 uL

Mix everything in the Nutator for 1-2 hrs. Then aliquate into 500 ul into an orange cap screw tubes and stored in -70 ⁰ C freezer. Cut the tip of 1000ul because the pre- Hyb is viscous

Formamide.

Aliquate 1ml of fomamide in 1ml orange screw cap tube and store in –70 .

Final Hybridization Mix.

Mix equal quantity of pre-hybridization and formamide for hybridization. This step you can do at the time of experiment.

50 % Dextran Sulfate ( 40ul–final Volume)

Add 20 gram of Dextran Sulfate (on Sridevi’s cabinet) to 10ml of dd water in a 50 ml conical tube. Add Dextran sulfate little by little because it will make clumps. Put the tube in the rocker/shaker. Add water below 37ml. Leave it in the rocker at least one day or two days. Then make up the volume to 40 ml, cover with tin foil and store it 4 degree celsius.

tRNA (100mg/mL)

Add 250ul of dd water to 25 mg of tRNA powder. (Usually we order 25mg in a vial)

Boeringer Block –10X

Dissolve 25 gms Block powder in 250 mls of1M Tris + 1.5M Nacl₂ pH 7.5.

Heat in microwave to 60 deg C. stir until it dissolves. Then aliquate 45 mls in 50 ml conical tubes and freeze in –80 freezer. It will come for 5 experiments.

—— —————

Day-1 Preparations

 

1. Turn on 80 deg. heat block
2. Turn on 55 deg oven for Hybridization

Solutions preparation.

 

1. 50mM Tris pH7.5 and 5mM EDTA ( for Proteinase K )

4.5 ml EDTA (0.5M)

22.5 ml. Tris pH 7.5 or pH 8.0 (1M)

423.0 ml ddH2O

Remove 1 ml of this solution to a clean eppendorf tube to make up the proteinase K   solution later. Warm the rest of the solution to 37 deg C in water bath in advance. Tightly seal to prevent contamination.

Proteinase K (This step should do before you incubate slides for proteinase k digestion).

Invitrogen proteinase k- cat # 25530-015.

Take 3 ug = 0.0030 gms and dissolve in 1 ml of Tris/EDTA solution. From that take 45ul into 450 ml of Tris/EDTA buffer.

2. Paraformaldehyde   solution. (4% in 1X PBS)

350 ml ddH2O

40 ml 10X PBS

heat liquid to approx. 60 deg. C in microwave.

add 16 grams of paraformaldehyde. mix on stir plate in hood

add one chip of NaOH to bring pH up to approx. 11.

let it dissolve and then bring back to room temp. (Can use ice if in a hurry)

Adjust pH with 60 ul of H2SO4 to close to pH 7.0. Check this with pH paper.

3. 0.2% Glycine in 1X PBS

50 ml of 10X PBS

450 ml ddH2O

Dissolve 1 gram of glycine in 500 ml 1X PBS solution

4. 1X PBS

50 ml of 10X PBS

• ddH2O

5. 0.2x SSC ( 2000mL)

Add 20ml of 20X SSC to 2000mL of DEPC water and place it in the 55 degree celsius oven overnight for tomorrow washes.

6. Xylenes in 2 glass dishes with micro spin bars. Keep it in the hood.
7. Ethanol series.

ETOH (95%) except for the first two which are 100% absolute	ddH2O	NaCl (5M)		time in solution
500	–		100 % ETOH	1 min.
500	–		100 % ETOH	1 min.
500	–		95 % ETOH	30 sec
446	39	14.7	85 % ETOH, plus 0.85% NaCl	30 sec
367	118	14.7	70 % ETOH, plus 0.85% NaCl	30 sec
263	223	14.7	50 % ETOH, plus 0.85% NaCl	30 sec
158	327	14.7	30 % ETOH, plus 0.85% NaCl	30 sec
–	485	14.7	0.85% NaCl	2 min.
			ddH2O	5 min.
			1XPBS(50ml PBS+450ml of water)	2 min.

 

Procedures

1. Mark slides with date and type of probe going tobe used. Put slides in metal rack (30 slides holders). Incubate in 100% xylenes for 10 min.
2. Again in 100% xylenes for 10 minutes.
3. Then follow the Ethanol series.

Series	time in solution
100 % ETOH	1 min.
100 % ETOH	1 min.
95 % ETOH	30 sec
85 % ETOH, plus 0.85% NaCl	30 sec
70 % ETOH, plus 0.85% NaCl	30 sec
50 % ETOH, plus 0.85% NaCl	30 sec
30 % ETOH, plus 0.85% NaCl	30 sec
0.85% NaCl	2 min.
ddH2O	5 min.
1XPBS(50ml PBS+450ml of water)	2 min.

4. Proteinase K Digestion .

Incubate for 30 minutes at 37 deg. C with occasional agitation.

Note: depending on the strength of the individual batch of Proteinase K you may have to titrate this to make sure you got it correct. If the tissue looks really chewed up you used too much for too long, if there is a very weak or no signalat the end of the in situ, one possibility is that you didn’t digest long enough. Unfortunately there are other places where things can go wrong to lead to the no signal result.

Stop proteinase K reaction with a 2 min. wash in 0.2% glycine solution in 1X PBS solution.

2min in 1X PBS – 2 washes

Incubate 10 minutes in 4% paraformaldehyde/1xPBS solution in hood.

5 min. in 1X PBS – 2 washes.

5. Ethanol series.

Start at 30 % ETOH, plus 0.85% NaCl up to 100% ETOH. 30 sec to 1 min each step. After final 100% ETOH wash (use fresh 100% ETOH for this final wash step) then remove rack and dry a bit on paper towels. remove excess ETOH by tapping bottom of metal rack well against paper towels.

6. Drying

Incubate for 30 min. in a vacuum drying apparatus. We use a vacuum oven with no heat on. Get the slides totally dry by pulling a vacuum.

Hybridization.

Pre- Hybridization preparation.

Thaw prehybridization mix and formamide (aliquate already in orange cap tube) from the –70 freezer. For 30 slides, I usually grab 6 tubes (500ul each) prehybridization mix and 4 (1ml each) formamide tubes.

Mix equal quantity of pre hyb and Formamide and leave on the nutator for 30 minutes.

Probe preparation.

Probe ingredients	1X(1 pair)	5x(enough for 4 pairs )
Dig labelled RNA	1ul	5ul
DEPC water	14ul	70ul
Formamide	15ul	75ul
Total	30ul

 

Mix DEPC water, formamide and probe. Denature at 80 degrees C for 2 minutes. Quick cool in ice water bath. Spin down and keep on ice. Amount of probe indicated is approximate, it is good to try 1 microliter per slide pair to start if you had a desent sized band of RNA after the transcription.

Then for each probe pair mix 170 ul of hyb mix for each 30 ul of probe/formamide mix. Mix well by inversion. This stuff is viscous and must be mixed well before applying to the slide pairs. try to invert in for several minutes to mix without adding bubbles to the solution. Use pipet tip to mix well

Hybridization mix	One slide pair	X
Pre Hyb/Formamide mix	170 ul
Probe	30 ul
Total	200 ul

Take a pair of slides. Add 100- 130 microliters Hybridization mixture to the top of each slide (200 – 260ul microliters per pair). Spread out across the dried tissue sections with the side of a clean micropipette tip. Use surface tension to avoid disturbing the tissue sections. Once all the tissue sections are wet. Hold the two slides upright so that the solution starts to fall to the bottom edge of the slide. Hold the two bottom edges of the slides touching each other at the raised white triangles (on the “probe-on-probe plus” slides). do this while resting the bottom of the slides on top of a piece of parafilm to prevent the probe from running off the slides. Slowly move the slides together creating a sandwich and allowing the probe to squeeze up between the slides, covering the tissue sectios without generating too many air bubbles. This takes a bit of practice. There is no need to seal the edges of the slide sandwich with rubber cement. Moist environment of hyb. Box will keep slides wet.

Change gloves between probes.

Place slide pairs on homemade plastic pipet rack inside tupperware box.

Seal tupperware with parafilm to make sure it stays moist.

Hybridize for Overnight to 36 hrs at 56 deg C. Use the most constant temp incubator you can find.

Note : Don’t forget the make 0.2X SSC ( about 2000ul) and keep it in 55 degree C. Keep 2 Glass staining dishes and slide metal rack at 55 deg C too.

 

Day 2- Washing steps

Washing with 0.2X SSC.

Separate slides by dipping in o.2X SSC. Change SSC between probes. Put slides on metal rack and keep it in 0.2X SSC at 55 deg C in an oven with rocking platform.

In Situ Step by Step- Day 2
WASHING STEPS	INCUBATION TIME AND TEMPERATURE	PREPARATION
0.2X SSC	60 min at 55 degree C in an oven with agitation	Prepare for 2000 ml 20 ml of 20x SSC 1980 ml of DEPC water
0.2X SSC	60 min at 55 degree C in an oven with agitaion
Boeringer Block	45 min with agitation at RT	45 ml of 10x stock( grab from –80 and thaw) 400 ml of DEPC water
TBNT	45 min with agitation at   RT	Prepare for 2500ml 25 gms of BSA 250 ml Tris/Nacl stock pH7.5 750 ml of 10% Triton X 2150 ml of DEPC water
1:1250 (antibody:TBNT ) Solution	Place slide in slide mailers five per mailer. Fill the mailer with 12ml of ab:TBNT soln.	Prepare for 2 batches(38ml in each) Total about 76ml. 38 ml of TBNT 31 ul of Dig oxigenin
TBNT	20 minutes with agitation
TBNT	20 minutes with agitation
TBNT	20 minutes with agitation
TBNT	20 minutes with agitation
100mM Tris pH 9.5; 150mM Nacl 50mM Mgcl2	15 Minutes with agitation	Prepare for 1500 ml 150 ml of 1M Tris/1.5Mnacl, 150 ml of 0.5M of Mgcl2(open in hood) 1200 ml of DEPC water
100mM Tris pH 9.5; 150mM Nacl 50mM Mgcl2	15 Minutes with agitation
100mM Tris pH 9.5; 150mM Nacl 50mM Mgcl2	15 Minutes with agitation	Make sure all the detergent has washed off.
Western blue	Overnight in dark.	Place slides in clean plastic slide mailers and fill these with Western blue from promega. Need about 12mls for each mailer.
Check under scope for development on next day morning.

 

Insitu – Final step

Stoping western blue reaction with TE (10mM Tris pH 7.5 &1mM EDTA pH8.0)

Make 500ml of TE for first wash.

1M Tris pH7.5    ———-              5 ml

0.5M EDTA pH8.0 ——-             1 ml

deionised water ———- –          494 ml

————–

Total                                                500 ml

—————-

Washing 1. wash with TE for 10 minutes

Washing 2. wash with deionised water for 5 minutes without agitation.

Mounting. Drain the excesss water on the paper towel. Then ,add 4 drops of Aqua poly mount on the slides and cover with coverslip.

 

 

 

IN SITU STOCK SOLUTIONS
STOCK NAME	COMPONENT	M.W.	GRAMS	Preparation and sterilization
20 X SSLC -1L	3 M Nacl	58.44	175.3 gms	DEPC treatment and   autoclave
pH 7.0	300 mM Na Citrate	294.1	88.2 grm
10X PBS – 1L	1.3 M Nacl	58.44	75.97 gms	DEPC treatment and   autoclave
pH 7.0	70 mM Na2HPo4	141.96	9.93 gms
	30 mM NaH2Po4	137.99	4.13 gms
0.5M EDTA – 1L	0.5 M EDTA	372.24	186.1gms	use NaoH to dissolve EDTA (about 18 gms)
p.H 8.0				DEPC treatment and   autoclave
0.5M Mgcl2 – 1L	0.5 M Mg cl2	203.3	101.65gm	DEPC treatment and   autoclave
5M Nacl -1L	5 M Nacl	58.44	292.2 gm	DEPC treatment and   autoclave
1M Tris/1.5M Nacl
pH 7.5	1M Tris HCl	157.6	157.60gms	initial pH reading was 4.15
1L	1.5 M Nacl	58.44	87.66gms	use NaoH pellet about 100 to bring pH 7.5
				autoclave . No DEPC treatment
1M Tris/1.5M Nacl
pH9.5	1M Tris free base	121.14	121.44 gms	Initial pH readings was 10.34
1L	1.5M Nacl	58.44	87.66 gms	Use HCL to bring down pH 9.5
				autoclave. No DEPC treatment
Denhards 50X	PVP		0.5 g
	Ficon400		0.5 g
	BSA (Fraction V)		0.5 g	bring up to 50 ml with DEPC water
· ·       DEPC Treatment:
Use 0.8 ml of DEPC to each 1 litre of solution and shake well with parafilm in the chemical hood
Then covered with Aluminium foil loosely for overnight. Make sure the rims are wet with the solution.

 

Chemicals for in situ hybridization

Name	Vendor	Cat. No.	Price($)
ApaL I	BioLabs	R0507S	58
Nco I	Promega	R6513	81
4-CORE® Buffer Pack (Buffers A, B, C, D)	Promega	R9921	10
Riboprobe® System – SP6	Promega	P1420	133
Riboprobe® System – T7	Promega	P1440	133
RNase free DNase	Promega	M6101	45
DIG RNA Labeling Mix	Roche	11277073910	146
Anti-Digoxigenin-AP	Roche	11093274910	200.00
Blocking Reagent	Roche	11096176001	114.00
Ficon 400	Sigma-Aldrich	F2637-5G	29.60
formamide	Sigma-Aldrich	F9037-100ML	44.70
PVP	Sigma-Aldrich	234257-5G	49.20
PVP	EMD	529504-100GM
BSA, Fraction V	EMD	2930	177.00
DEPC	EMD	3660	154.00 × 2
Xylenes	Fisher	X3P-1GAL	116.46
Western Blue	Fisher	PR-S3841	48.46 × 2
Dextran Sulfate	Fisher	BP1585-100	246.86
Glycine	Sigma	G7126-100g	20.30
H2SO4
Proteinase K	Invitrogen	25530-015	96.00
tRNA	Invitrogen	15401-029	127.00
paraformaldehyde	Ted Pella, Inc	18501	22.90
100% ethanol	pharmci-aaper	111ACS200
Aqua poly/Mount	Polysciences, Inc.	18606-20	60.00
Triton X-100	Fisher	BP151500	46.12
0.375% Elmer’s Carpenter’s Wood Glue

Facilities for in situ hybridization

Name	Vendor	Cat. No.	Price($)
Probe Microscope Slides	Fisher	22-230-900	80.98
Oven (for hybridization)	Precision, Thelco		About 1500
Oven (for paraffin)	Curtin Matheson Scientific, Inc.
Microwave oven	BioWavePro, PELCO
Microscope	Nikon ECLIPSE E600
Tissue Imbedding Capsules	Fisher	15-182-218
Paraffin	SPI	2846A-AB
Boat
Plastic frames
Megohms-CM 75 II	MyronL Company	B2-VW-E3452	220.28
Rotator	Barnstead Lab-Line		About 800
Stir plate	Nuova ll Thermolyne
Coverslips	Fisher	12-543B	33.87
Coverslips	Fisher	12-543D	33.67
Coverslips	BioExpress	2935-246-G
Coverslips	VWR	48382-139	139.89
RNase free tips
RNase free 1.5ml tubes
Clat Adams Nutator	VWR	15172-203	633.89
Glass cylinders – 1000ml	VWR	89000-276	72.24 × 4
Glass cylinders –500ml	VWR	89000-274	66.45 × 4
Very small size stir bar	VWR	58948-411	6.92 × 2
Small size stir bar	VWR	58948-138	8.69 × 10
Medium size stir bar	VWR	58948-150	10.80 × 10
Large size stir bar	VWR	58948-171	13.02 × 10
Very large size stir bar	VWR	58948-309	13.37 × 5
Razer blades	VWR	55411-055	27.59
pH indicator strips	VWR	60772-001	33.69
Glass flasks – 3000ml	OnlineScienceMall	FLA206	42.95 × 6
Glass dishes	Ted Pella, Inc	21054	69.90× 7
Metal rack	Ted Pella, Inc	21072	48.90× 2
Slide mailer	VWR	82003-430	14.81
Plastic chamber	Walmart		× 2
Plastic box	Walmart		× 30
Big plastic containing holding 20L	Walmart		× 1