Reagents

Trouble shooting

A. Prehybridization

1. Take slides out from fridge

2. Dewax in Xylene 2 X 10'
3. 100 % Ethanol 2'
4. 100 % Ethanol 2 X 1'
5. 95 %, 80 %, 70 %, 50 %, 30 % 1'@
6. Saline (0.83 % NaCl) 5'
7. PBS 5'
8. 4 % Paraformaldehyde in PBS (fresh) 20'
9. PBS 2 X 5'
10. PK (20 mg/ml in TE) 5'

*Critical step! For 9.5 dpc embryos use a 2/5 P.K. solution (8 ug/ml) and keep the timing to exactly five minutes. Failure results in degradation of the sample PK conditions will also change according to batch and source.

11. PBS 5'
12. 4 % Paraformaldehyde 20'
13. DDW Quick
14. Triethanolamine (TEA)+ stir bar
15. Acetic anhydride (0.5 ml/200 ml TEA) 10'
16. PBS 5'
17. Saline 5'
18. 30 %, 50 %, 70 %, 80 %, 95 % 1'@

19. 100 % Ethanol 2 X 2'
20. Air dry

*To leave overnight, seal slides in an air tight jar with lots of silica gel and store at -20 ^oC.

B. Hybridization

Before starting, book the hybridisation oven for overnight at 50 ^oC and put the hybridisation jar into it so it can pre-warm.

1. Wash cover slip with 100 % Ethanol and cut it to appropriate size.
2. Heat probe at 80 ^oC for 2 mins.
3. Quick spin the tube and SpeedVac the probes for 1/2 to 1 min.
4. Apply 20 ul-40 ul of probe to section.

*Apply probe to one side of the section and place on the coverslip carefully so that bubbles are avoided. When bubbles do appear under the coverslip the squeeze them out by pressing gently on the glass with a hyperdermic needle or forceps. (Little bubbles don't matter too much).
5. Gently put cover slip on without trapping any bubbles.
6. Put slides horizontally in slide box with tissue paper soaked with 50% formamide, 5X SSC and then put the box in a large sealed jar.
7. Incubater overnight at 50 ^oC.

It is as well to pre-warm a post-hybridisation jar for tomorrow with a bottle of 5XSSC for the first step in post-hybridisation. (add the DTT tomorrow).

*Book the shaking waterbath for tomorrow, at 60 ^oC and also make sure there is enough deionised formamide.

 

*Also put enough 5X SSC in the hybridisation oven for the first step of post-hybridisation.

C. Post-Hybridization

Take out the deionised formamide from the fridge and make sure that crystals any crystals have dissolved before you start to aliquot it.

1. Remove slides into glass rack and put in 5X SSC, 10mM DTT 50 ^oC,30-60' (Shaking waterbath.)
2. 50 % formamide, 2X SSC, 20mM DTT 60 ^oC, 30'
3. NTE 3X 10'
4. RNase (in NTE) 37 ^oC, 45'

*Use good RNase set aside for in situ. (It is actually fragile so keep it on ice and don't use RNA pipettes!!)

5. 50 % formamide, 2X SSC, 20mM DTT 60 ^oC, 1 hr
6. 2X SSC 15'
7. 0.1X SSC 15' (15' is OK but an hour is best.)
8. 30 %,60 %,80 %,95 % + NH4Ac 1'@
9. 100 % Ethanol 2 X 2'
10. Air dry
11. Put in stainless steel rack and ready for emulsion dipping.

*If leaving the slides for some time before dipping then wrap them in foil and put them in the dry box.

 

D. Emulsion dipping

 

1. Under dark condition, place 8 ml emulsion shreds in a universal tube and melt (~20 minutes) at 42 ^oC

2. Transfer 8 ml 2 % glycerol (pre-warmed at 42 ^oC) into the molten emulsion and roll the tube gently (do not create bubbles in the emulsion)

3. Transfer emulsion to the slide mailer with a wide-mouth pipette

4. Check whether emulsion is bubble-free by dipping a clean slide and examining it for even coating

5. Repeat until bubbles are removed

6. Dip the experimental slides, allowing each to drain vertically for 2 seconds

7. Wiping the back of the slide and placing it horizontally to let them dry (this takes at least half an hour)

8. Transfer the slides to a slide box containing a sachet of desiccant

9. Seal the box with tape and place it at 4 ^oC. It is important that the slides are completely dry during exposure

 

E. Slide development

 

1. Remove the slide box from 4 ^oC and warm to room temperature (>1 hour)

2. Develop slides as follows:

Developer	2 minutes
Stop	1 minute
Fixer	2 minutes
Cold tap water	3X 5 minutes

 

F. Counterstain

 

1. Alcohol dehydration (50 %, 70 %, 95 % and 100 %) is required before air dry or put into dry box. Before H&E staining, slides were placed in tap water for one minute. (Optional)

2. Transfer slides to stainless steel rack since dry box plastic rack dissolves in toluene

3. Haematoxylin 45 seconds

4. Wash slides in cold tap water (from dark room) gently several times until all haematoxylin is removed

5. Dip in acid ethanol quickly (1 % HCl, 70 % ethanol)

6. Transfer to tap water several times gently

7. Scott's tap water

8. Tap water => observe the colour intensity

9. Eosin 15 seconds

10. Dehydration - ascending alcohols to 100 % ethanol (rapid changes)

70 % EtOH	1 second
95 % EtOH	1 second
95 % EtOH	destain eosin to pink
100 % EtOH	2X 2 minutes
100 % EtOH	5 minutes

11. Clearing with 2X 5 minutes in toluene

12. Mounting with mounting medium e.g. Permount