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Gaido Group Protocols

Part 1 of 3 (next)

GUDMAP – SOP “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

DAY 1:
Acetylation of frozen sections cut at 10µm
Assembly of hybridization chambers
Daily Solutions
Starting the run
Steps (Prehyb, hyb, post-hyb, probe detection reactions)

 

DAY 2:
Disassemble hyb chambers
Clean-up
Shut-down

 

The Tecan script was supplied by Tecan.

Unless otherwise stated the following protocol requires ultra-pure water (Milli-Q).

For list of solutions & chemicals see appendix.

Essential Practices

  • Always wear gloves and use RNase-free reagents and materials.
  • Always protect the color reagent (NBT, BCIP) and other sensitive chemicals from direct light. As most color substrates are light sensitive they lose their sensitivity as they are exposed to light.

 

DAY 1

Tissue Preparation for ISH in Freedom Evo (Acetylation)
Make 200mL of each solution unless otherwise noted.

1.

Isolate tissue.

2.

Freeze tissue in OCT by placing tissue in plastic molds with OCT, arranging the tissue as desired, and freezing the mold on dry ice.

3.

Store at -80°C

4.

Section tissue into 10μm sections.  Be sure to use “precleaned Superfrost plus” slides (Fisher 12-550-15).  Label slides with a pencil.  Store in a plastic bag with desiccant at -20°C, overnight (or up to 3 weeks).

When ready to acetylate:
Heat fix slides at 50°C for 2 minutes (or before using).

5.

Fix:  Fix sections in 4% PFA / PBS for 10 min RT (make up fresh every time). 
(40 mL PFA/160 mL PBS DEPC H2O.)

6.

Rinse in 1X DEPC PBS for 3 minutes, 3X.

7.

Acetylation:  In a container, on a stirrer, combine: Tissue Tek holds 200mL

 

 

H2O (DEPC)
Triethanolamine (dissolve in H2O & HCl before hand)
37% HCl 10mM final

 

 

200mL
2.66mL
350µl

 

Acetic Anhydride 0.5% final 

 

750µl (add last)

 

Immerse sections in the above solution for 10 min room temperature (add 750µl acetic anhydride, incubate at room temperature for 5 minutes, after 5 minutes add another 750µl of acetic anhydride and incubate at room temperature for 5 minutes.)  Acetic anhydride is only good for 5 minutes, adding 2x @ 750µl each gives a final concentration of 0.5%.
KEEP ACETIC STEP ON STIRRER AT ALL TIMES DURING INCUBATION!

 

  8. 

Rinse in DEPC PBS for 3 minutes, 3X

  9.

Series dehydration (1-2 minutes for each, see below steps 9a – 9f)

 


 

DEHYDRATION STEPS

9a. 70% EtOH DEPC H2O
9b. 80% EtOH DEPC H2O
9c. 95% EtOH DEPC H2O
9d. 95% EtOH DEPC H2O
9e. 100% EtOH DEPC H2O
9f. 100% EtOH DEPC H2O

 

10.

Air dry and store in sealed boxes at -80°C or use immediately for ISH. (<1 month storage, 3-5% degradation per month).

 

  NOTE:  EtOH solutions are only good for up to 2 weeks after diluting, make fresh every two weeks.

 

Assembly of Hybridization chambers

  1. Thaw the desired number of acetylated slides from the -80°C freezer, if acetylated before hand, in a 37°C incubator for about five minutes.  If slides were acetylated the same day as the run then slides do not need to thaw at 37°C.
  2. Make sure the area is RNase free and collect all needed equipment and tools.   Including: assembly platforms, aluminum assembly frames, assembly clips, Tecan spacers, glass back plates, acetylated slides and scissors. 
  3. Begin placing aluminum assembly frames onto the assembly platforms in their respective grooves.  Begin laying the slides down into the aluminum assembly frame with the label toward the centre of the workbench facing upward.  Make sure slides are against the top of the assembly frame. 
  4. Place the Tecan spacers on top of the slides.  It is important for the spacers to be placed within the frame with equal overhang to prevent damage to slides, and to ensure proper solution flow through the hybridization chamber. 
  5. Carefully lay the back plate on top of the slide and spacer, against the top of the assembly frame, with the etched reservoir facing downward onto the label. 
  6. Fasten the back plate to the assembly frame with the assembly clip.  To clip the back plate to the frame, hold the back plate onto frame with slight pressure with one hand and hook one end of clip and pull over and down with the other hand.  An audible click should be heard when done correctly.  Two assembly clips per aluminum assembly frame are used.  One is placed just below the etched reservoir and the other about a half-inch above the bottom of the glass back plate. 
  7. With a pair of scissors, cut the overhang of the spacers off by carefully cutting along the top of the assembly frame and along the bottom of the slide.  Be careful not to cut the slide with scissors, may cause slide to crack.
  8. Place the hyb chambers in their respective position on the platform.


Group Description